TW202142566A - Anti-ox40 antibodies and methods of use thereof - Google Patents

Abstract

The present disclosure provides antibodies that specifically bind to human OX40 receptor (OX40) and compositions comprising such antibodies. In a specific aspect, the antibodies specifically bind to human OX40 and modulate OX40 activity, e.g., enhance, activate, or induce OX40 activity, or reduce, deactivate, or inhibit OX40 activity. The present disclosure also provides methods for treating disorders, such as cancer, by administering an antibody that specifically binds to human OX40 and modulates OX40 activity, e.g., enhances, activates, or induces OX40 activity. Also provided are methods for treating autoimmune or inflammatory diseases or disorders, by administering an antibody that specifically binds to human OX40 and modulates OX40 activity, e.g., reduces, deactivates, or inhibits OX40 activity.

Description

Anti-OX40 antibodies and methods of use

[Sequence Listing]

This application includes a sequence listing, which has been electronically submitted in ASCII format and incorporated herein by reference in its full text (the ASCII copy created on May 5, 2016 is named 3617_003TW04_SL.txt and has a size of 120,927 bits. ).

The present disclosure relates to antibodies that specifically bind to the human OX40 receptor ("OX40"), compositions including such antibodies, and methods for producing and using antibodies that specifically bind to OX40.

The contribution of innate and acquired immune responses in the control of human tumor growth is well characterized (Vesely MD et al. (2011) Annu Rev Immunol 29:235-271). As a result, antibody-based strategies have emerged, which are aimed at enhancing T cell responses for cancer treatment purposes, such as targeting stimulatory receptors expressed by T cells with elicitation antibodies, or targeting inhibition with functional antagonists Sex Body (Mellman I et al., (2011) Nature 480:480-489). Antibody-mediated agonist and antagonist approaches have shown preclinical activity, and recently have shown clinical activity. OX40 receptor (also known as OX40, CD134, TNFRSF4, TXGP1L, ACT35, and ACT-4) is an important stimulatory receptor system that regulates the function of T cells, natural killer T (NKT) cells, and NK cells (Sugamura K et al., (2004) Nat Rev Immunol 4:420-431). OX40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF), and it can adjust important immune functions through OX40 signaling.

After T-cell receptor (TCR) stimulation by professional antigen-presenting cells (APC) displaying MHC class I or II molecules loaded with homologous peptides, OX40 can be upregulated by antigen-specific T cells (Sugamura K et al., ( 2004) Review of Nature Immunology 4:420-431). When mature, APCs (such as dendritic cells (DC)) upregulate stimulatory B7 family members (such as CD80 and CD86), together with auxiliary costimulatory molecules (including OX40 ligand (OX40L)), which help shape T The dynamics and size of the cellular immune response, along with effective memory cell differentiation. Obviously, other cell types can also exhibit constitutive and/or inducible levels of OX40L, such as B cells, vascular endothelial cells, mast cells, and in some cases activated T cells (Soroosh (Soroosh) P Et al. (2006) J Immunol 176: 5975-5987). It is believed that OX40:OX40L co-binding drives higher order clustering of receptor trimers and subsequent signal transduction (Compaan DM et al., (2006) conclusion Structure (Structure) 14: 1321-1330).

The OX40 expression of T cells in the tumor microenvironment has been observed in murine and human tumor tissues (Bulliard Y et al., (2014) Immunol Cell Biol 92:475- 480 and Piconese S et al. (2014) Hepatology 60:1494-1507). Compared with the conventional T cell population, the intratumoral population of regulatory T cells (Treg) highly express OX40, which is attributed to their proliferative state (Waight JD et al., (2015) J Immunol 194:878-882 and Bulliard Y et al. (2014) Immunol Cell Biol 92:475-480). Early studies have shown that OX40-evoked antibodies can cause tumor rejection in mouse models (Weinberg AD et al. (2000) Journal of Immunology 164:2160-2169 and Picansi S et al., (2008) J Exp Med 205:825-839). It has also been shown that mouse antibodies stimulated by human OX40 messaging enhance the immune function of cancer patients (Curti BD et al. (2013) Cancer Res 73: 7189-7198).

The interaction between OX40 and OX40L is also related to the immune response in inflammatory and autoimmune diseases and disorders, including asthma/atopic, encephalomyelitis, rheumatoid arthritis, colitis/inflammatory bowel disease, transplantation Animal versus host disease (e.g., transplant rejection), diabetes in non-obese diabetic mice, and mouse models of arteriosclerosis (Croft M et al., (2009) Immunological Review (Immunol Rev) 229(1): 173-191, and references cited therein). The reduction in symptoms associated with diseases and disorders has been reported in OX40- and OX40L-deficient mice, mice receiving anti-OX40 liposomes loaded with cytostatic drugs, and in which the interaction of OX40 and OX40L is blocked by anti-OX40L In mice blocked by antibodies or recombinant OX40 fused to the Fc fragment of human immunoglobulin (Croft M et al.; Bute EPJ et al. (2005) Arthritis Res Ther) 7 : R604-615; Weinberg AD et al. (1999) Journal of Immunology 162: 1818-1826). It has also been shown that treatment with a blocked anti-OX40L antibody can inhibit Th2 inflammation in a rhesus asthma model (Croft M et al.; Seshasayee D et al. (2007) J Clin Invest 117: 3868- 3878). Additionally, the polymorphism of OX40L has been associated with lupus (Croft M et al.).

In view of the role of human OX40 in regulating the immune response, antibodies that specifically bind to OX40 and the use of these antibodies to regulate the activity of OX40 are provided here.

In one aspect, provided herein are antibodies that specifically bind to OX40 (e.g., human OX40).

In one embodiment, the antibody that specifically binds to OX40 includes: the heavy chain variable region (VH) CDR1 of the VH CDR1 contained in SEQ ID NO: 16 and the VH CDR2 of the VH CDR2 contained in SEQ ID NO: 16 , VH CDR3 contained in SEQ ID NO: 16 VH CDR3, the light chain variable region (VL) CDR1 of the VL CDR1 contained in SEQ ID NO: 15, the VL CDR2 of the VL CDR2 contained in SEQ ID NO: 15 and the VL CDR2 contained in SEQ ID NO: 15 VL CDR3 of VL CDR3, wherein each is defined according to the Kabat definition, Chothia definition, combination of Kabat definition and Chothia definition of CDR, IMGT numbering system, AbM definition, or contact definition (contact definition) CDR.

In one embodiment, the antibody that specifically binds to OX40 includes: (a) a heavy chain variable region including the heavy chain complementarity determining region 1 (CDR1), the heavy chain CDR1 including the amino group of GSAMH (SEQ ID NO: 4) Acid sequence; heavy chain CDR2, which includes the amino acid sequence of RIRSKANSYATAYAASVKG (SEQ ID NO: 5); and heavy chain CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); and (b) including light The light chain variable region of the chain CDR1, the light chain CDR1 includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); the light chain CDR2, which includes the amino acid sequence of LGSNRAS (SEQ ID NO: 2); and the light chain CDR1 Chain CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3).

In one embodiment, an antibody that specifically binds to OX40 includes: (a) a heavy chain variable region including a heavy chain CDR1 that includes the amino acid sequence of GFTFSGSA (SEQ ID NO:47); a heavy chain CDR2 , Which includes the amino acid sequence of IRSKANSYAT (SEQ ID NO: 48); and the heavy chain CDR3, which includes the amino acid sequence of TSGIYDSSGYDY (SEQ ID NO: 49); and (b) includes the light chain CDR1 The light chain variable region, the light chain CDR1 includes the amino acid sequence of QSLLHSNGYNY (SEQ ID NO: 44); the light chain CDR2, which includes the amino acid sequence of LGS (SEQ ID NO: 45); and the light chain CDR3, It includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 46).

In one embodiment, the antibody includes a heavy chain variable region with a human or human-derived framework region.

In one embodiment, the antibody includes a heavy chain variable framework region derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence includes IGHV3-73*01 (SEQ ID NO: 19).

In one embodiment, the antibody includes a light chain variable sequence with human or human-derived framework regions.

In one embodiment, the antibody includes a light chain variable framework region derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence includes IGKV2-28*01 (SEQ ID NO: 18).

In one embodiment, the antibody includes a heavy chain variable region sequence, which includes the amino acid sequence of SEQ ID NO:16.

In one embodiment, the antibody includes a heavy chain sequence, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 51, and 52. In one embodiment, the antibody includes a heavy chain sequence, which includes an amino acid sequence selected from the group consisting of: SEQ ID NO: 60-63.

In one embodiment, the antibody includes a light chain variable region Sequence, which includes the amino acid sequence of SEQ ID NO:15.

In one embodiment, the antibody includes a light chain sequence, which includes the amino acid sequence of SEQ ID NO:20.

In one embodiment, the antibody that specifically binds to OX40 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NO:16.

In one embodiment, the antibody that specifically binds to OX40 includes a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes the amino acid sequence of SEQ ID NO:15.

In one embodiment, the antibody that specifically binds to OX40 includes: a heavy chain variable region, which includes the amino acid sequence of SEQ ID NO: 16; and a light chain variable region, which includes the amino acid sequence of SEQ ID NO: 15 Acid sequence.

In one embodiment, the antibody includes: a heavy chain, which includes the amino acid sequence of SEQ ID NO: 21; and a light chain, which includes the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody includes: a heavy chain including the amino acid sequence of SEQ ID NO: 60; and a light chain including the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the antibody includes: a heavy chain, which includes the amino acid sequence of SEQ ID NO: 23; and a light chain, which includes the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody includes: a heavy chain, which includes the amino acid sequence of SEQ ID NO: 61; and a light chain, which includes the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the antibody includes: a heavy chain, which Including the amino acid sequence of SEQ ID NO: 51 or 52; and the light chain, which includes the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody includes: a heavy chain, which includes the amino acid sequence of SEQ ID NO: 62 or 63; and a light chain, which includes the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the antibody includes a heavy chain and/or light chain constant region. In one embodiment, the heavy chain constant region is selected from the group consisting of: human immunoglobulins IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In one embodiment, the IgG ₁ is a non-fucosylated IgG ₁ . In one embodiment, the amino acid sequence of _{IgG 1 includes the N297A mutation.} In one embodiment, _{the amino acid sequence of IgG 1} includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In one embodiment, the amino acid sequence of _{IgG 1 includes the N297Q mutation.} In one embodiment, the amino acid sequence of _{IgG 4 includes the S228P mutation.} In one embodiment, the amino acid sequence of _{IgG 2 includes a C127S mutation.} In one embodiment, the heavy chain constant region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-37, 53-54, and 64-71. In one embodiment, the light chain constant region is selected from the group consisting of: human immunoglobulins IgGκ and IgGλ.

In one embodiment, the antibody is a human antibody.

In one embodiment, the antibody that specifically binds to OX40 binds to the same epitope of human OX40 as the following antibody, the antibody includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid sequence of RIRSKANSYATAYAASVKG (SEQ ID NO: 5); VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes RSSQSLLHSNGYNYLD (SEQ ID NO: 1 ); VL CDR2, which includes the amino acid sequence of LGSNRAS (SEQ ID NO: 2); and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3). In one embodiment, the antibody that specifically binds to OX40 binds to the same epitope of human OX40 as the following antibody, the antibody includes: VH CDR1, which includes the amino acid sequence of GFTFSGSA (SEQ ID NO:47); VH CDR2, which Including the amino acid sequence of IRSKANSYAT (SEQ ID NO: 48); VH CDR3, which includes the amino acid sequence of TSGIYDSSGYDY (SEQ ID NO: 49); VL CDR1, which includes the amino acid sequence of QSLLHSNGYNY (SEQ ID NO: 44) Acid sequence; VL CDR2, which includes the amino acid sequence of LGS (SEQ ID NO: 45); and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 46). In one embodiment, an antibody that specifically binds to OX40 binds to the same epitope of human OX40 as an antibody that includes: a heavy chain variable region, which includes the amino acid sequence of SEQ ID NO: 16; and a light chain The variable region includes the amino acid sequence of SEQ ID NO:15.

In one embodiment, the antibody system is agonistic. In one embodiment, the antibody initiates, enhances or induces the activity of human OX40. In one embodiment, the antibody induces CD4+ T cell proliferation. In one embodiment, the CD4+ T cell proliferation line antibody concentration is Basically an incremental function. In one embodiment, CD4+ T cell proliferation exhibits a sigmoidal dose response curve. In one embodiment, the antibody induces the production of IL-2, TNFα, IFNγ, IL-4, IL-10, IL-13, or a combination thereof by anti-CD3-stimulated T cells. In one embodiment, the antibody induces TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-4, IL-10, IL-13 by anti-CD3-stimulated peripheral blood mononuclear cells (PBMC) , Or a combination thereof. In one embodiment, the antibody induces the production of TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13 by anti-CD3-stimulated PBMC, wherein TNFα, TNFβ, IFNγ, The production of GM-CSF, IL-2, IL-10, or IL-13 is a substantially increasing function of antibody concentration. In one embodiment, the antibody induces the production of TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13 by anti-CD3-stimulated PBMC, wherein TNFα, TNFβ, IFNγ, The production of GM-CSF, IL-2, IL-10, or IL-13 showed a sigmoidal dose response curve. In one embodiment, the antibody induces IL-2 production by SEA-stimulated T cells and inhibits IL-10 production by SEA-stimulated T cells. In one embodiment, the antibody induces IL-2 production by SEA-stimulated peripheral blood mononuclear cells (PBMC) and inhibits IL-2 production by SEA-stimulated PBMC. In one embodiment, the antibody induces IL-2 production by SEA-stimulated PBMC, where IL-2 production is a substantially increasing function of antibody concentration. In one embodiment, the antibody induces the production of IL-2 by SEA-stimulated PBMC, wherein the production of IL-2 shows a sigmoidal dose response curve.

In one embodiment, the antibody alleviates the suppression of effector T cells by regulatory T cells.

In one embodiment, the antibody induces the production of IL-2 by co-culture of effector T cells and regulatory T cells and inhibits the production of IL-10 by co-culture of effector T cells and regulatory T cells.

In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein the antibody is similar to Staphylococcal Enterotoxin A (SEA) (for example, 100ng/ml) In combination, when stimulated at 37° C., 5% CO ₂ , and 97% humidity, for example, 5 days later, the production of IL-2 in PBMC is induced, such as by electrochemiluminescence (e.g., human TH1/TH2 10- Plex tissue culture kit (kit) (Meso Discovery Company)), where IL-2 production is measured in, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and Substantially increasing antibody concentration between 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml function. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein the antibody is similar to Staphylococcal Enterotoxin A (SEA) (for example, 100ng/ml) In combination, when stimulated at 37° C., 5% CO ₂ , and 97% humidity, for example, 5 days later, the production of IL-2 in PBMC is induced, such as by electrochemiluminescence (e.g., human TH1/TH2 10- Plex tissue culture kit (Meso Discovery Company)), when the anti-OX40 antibody concentration is, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, When between 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml, the production of IL-2 shows an S-shaped dose response curve , As assessed in, for example, an assay that includes the following steps: (a) the antibody in the absence or presence of different concentrations (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and for example at 100ng / SEA ml of such culture PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example, at e.g. 37 ℃, 5% CO _2, and 97% humidity; and (b) collecting the supernatant was clarified And measure the IL-2 titer by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein the antibody is similar to Staphylococcal Enterotoxin A (SEA) (for example, 100ng/ml) In combination, when stimulated at 37°C, 5% CO ₂ , and 97% humidity for example for 5 days, the production of IL-2 in PBMC is induced, for example, by electrochemiluminescence (for example, human TH1/TH2 10- As measured by Plex tissue culture kit (Meso Discovery Inc.), the IL-2 production in the presence of 4 μg/ml antibody was greater than that in the presence of 0.032 μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein when the antibody is bound to the plate, the anti-CD3 antibody bound to the plate (for example, 0.8 μg /ml) in combination, when stimulated at 37°C and 5% CO ₂ for 4 days, for example, one or more cytokines (eg, TNFα, TNFβ, IFNγ, GM-CSF, IL) are induced in PBMC or T cells. -2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) or non-human primates (NHP ) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) The production line is for example 0.7μg/ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml. An incremental function, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g., 0.8 μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50 μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) in the presence of the antibody of the present invention bound to the plate, for example at 37° C. and 5% CO ₂ Cultivating the PBMCs for, for example, 4 days; and (b) collecting the supernatant and performing electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company)) or non-human primates (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) produce. In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein when the antibody is bound to the plate, the anti-CD3 antibody bound to the plate (for example, 0.8 μg /ml) in combination, when stimulated at 37°C and 5% CO ₂ for 4 days, for example, one or more cytokines (eg, TNFα, TNFβ, IFNγ, GM-CSF, IL) are induced in PBMC or T cells. -2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) or non-human primates (NHP ) V-Plex Assay Kit (Meso Discovery Company)), where the concentration of anti-OX40 antibody is, for example, 0.7μg/ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ ml, or between 6.3μg/ml and 50μg/ml, the production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) shows S-shaped dose-response curve, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody (e.g., 0.8 μg/ml) bound on the plate and different concentrations (e.g., 0, 0.3, 1, 3, 6 , 12, 25, and 50 μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) in the presence of the antibody of the invention bound to the plate, for example at 37° C. and 5% CO ₂ Culture the PBMCs for 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primates). Animal (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) The production.

In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) the amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein the antibody increases the proliferation of CD4+ T cells, wherein the CD4+ T cell proliferation line is, for example, 0.2 μg/ml And 20 μg/ml, or between 2 μg/ml and 20 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) for example, at 37° C. with, for example, 10 μM carboxyfluorescein Fluorescein Acetyl Acetate Succinimidyl Acetate (CFSE) is used to label, for example, enriched CD4+ T cells for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of, for example, plate-bound anti-CD3 antibody and Different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound antibodies described herein, stimulate the cells (e.g., in wells _{at 37°C and 5% CO 2).} 10 ⁵ cells); and (c) on, for example, day 4, cell staining with, for example, an anti-CD4 antibody and checking CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells by means of flow cytometry. In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein the antibody increases the proliferation of CD4+ T cells, wherein when the concentration of the anti-OX40 antibody is, for example, 0.2 μg/ml And 20μg/ml, or between 2μg/ml and 20μg/ml, CD4+ T cell proliferation shows a sigmoidal dose response curve, as evaluated in, for example, an assay that includes the following steps: (a) for example, 10 μM at 37°C Carboxyfluorescein acetoacetate succinimidyl acetate (CFSE) labels, for example, the enriched CD4+ T cells for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 Antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound antibodies described herein, stimulate the cells at, for example, _{37° C. and 5% CO 2 (e.g.,} in the bore ¹⁰⁵ cells); and (c), for example, on day 4, for example, anti-CD4 antibody cell staining and by means of flow cytometry by e.g. measuring low CFSE percentage of CD4 + cells is used to check the CD4 + T cell proliferation .

In one embodiment, the antibody that specifically binds to OX40 includes: VH CDR1, which includes the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid of RIRSKANSYATAYAASVKG (SEQ ID NO: 5) Sequence; VH CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); VL CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL CDR2, which includes LGSNRAS (SEQ ID NO : 2) amino acid sequence; and VL CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3), wherein when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml This antibody leads to a greater increase in CD4+ T cell proliferation, as assessed in, for example, an assay that includes the following steps: (a) Use, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37°C For example, the enriched CD4+ T cells are labeled for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20μg / ml) plates for example, antibodies described herein bind to, stimulate the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c) in e.g. On day 4, cells are stained with, for example, an anti-CD4 antibody and CD4+ T cell proliferation is checked by, for example, measuring the percentage of CFSE low CD4+ cells by means of flow cytometry.

In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH210-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or A substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) last for example 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Scale Discovery)), where the antibody is at 4 μg/ml The production of IL-2 is greater in the presence of the antibody than in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is at, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20 μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in, for example, an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody ( For example, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and 100ng/ml SEA, for example 37°C, 5% CO ₂ , and 97% humidity to cultivate the PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. electrochemiluminescence (e.g., a human TH1 / TH2 10-Plex tissue culture kit (meso found Company)) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is at, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml , Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) last for example 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is at, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) last for example 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) for 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) for 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is at, for example, 0.032 μg/ ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, Or a substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)). In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the concentration of anti-OX40 antibody is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or Between 0.8 μg/ml and 4 μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100ng/ml SEA, at 37°C, 5% CO2, and 97% humidity for example, culture the PBMC (e.g. , in the hole ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. electrochemiluminescence (e.g., a human TH1 / TH2 10-Plex tissue culture kit (meso found Corporation) ) Measure the titer of IL-2.

In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml), _{induces e.g. PBMC after stimulation at e.g. 37°C, 5% CO 2} and 97% humidity for e.g. 5 days The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the ratio in the presence of 4 μg/ml antibody The production of IL-2 was greater in the presence of 0.032μg/ml antibody. The production of IL-2 can be evaluated, for example, in an assay that includes the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ ml), and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) The clear supernatant is collected and the IL-2 titer is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, The production of TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) shows a sigmoidal dose response curve, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 bound to the plate Antibody (e.g., 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform, for example, electrochemiluminescence ( For example, the human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or the non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (e.g. , TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the anti-OX40 antibody concentration is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml And 50 μg/ml, or between 6.3 μg/ml and 50 μg/ml, as a substantially increasing function of the antibody concentration, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate (e.g. , 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml ) The plate-bound antibody of the present invention _{is cultured at 37°C and 5% CO 2} for example for 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production. In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 domain, wherein the anti-CD3 antibody, when combined with the plate, the plate binding (e.g., 0.8μg / ml) in combination, for example, 37 [deg.] C when 5% CO ₂ and the continuity of stimulation induced by e.g. PBMC or 4 days e.g. The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in T cells, such as by, for example, electrochemiluminescence (for example, human TH1 /TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), when the concentration of anti-OX40 antibody is, for example, 0.7 μg /ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ , IFNγ, GM-CSF, IL-2, IL-10, or IL-13) show a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (E.g. 0.8μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg /ml) plate-bound antibody of the present invention _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g. , Human TH1/TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) production.

In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 70% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 70% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 75% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 75% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 85% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 85% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as For evaluation in an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example, for 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay comprising the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidate (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, such as Evaluate, for example, in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C for, for example, 7 minutes (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the cells were stained by Flow cytometry checks CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve , As evaluated in, for example, an assay including the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate described herein antibody binding, 37 [deg.] C in the case of, for example, 5% CO _2, and stimulated the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody e.g. And by means of flow cytometry, CD4+ T cell proliferation can be checked by, for example, measuring the percentage of CFSE low CD4+ cells.

In one embodiment, the antibody includes a VL domain having at least 98% identity with the amino acid sequence of SEQ ID NO: 15 and a VH structure having at least 98% identity with the amino acid sequence of SEQ ID NO: 16 Domain, where the antibody results in a greater increase in CD4+ T cell proliferation when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ ml of, for example, plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, at 37°C and 5% CO ₂ for example (E.g., 10 ⁵ cells in the well); and (c) on, e.g., day 4, cell staining with e.g. anti-CD4 antibody and by means of flow cytometry by e.g. measuring CFSE low CD4+ cells Percentage to check CD4+ T cell proliferation.

In one embodiment, the antibody includes a human immunoglobulin IgG ₁ heavy chain constant region, wherein _{the amino acid sequence of the IgG 1} heavy chain constant region includes a mutation selected from the group consisting of: N297A, N297Q , D265A, and combinations thereof. In one embodiment, the antibody includes an IgG ₁ heavy chain constant region, wherein _{the amino acid sequence of the IgG 1} heavy chain constant region includes a mutation selected from the group consisting of: D265A, P329A, and combinations thereof .

In one embodiment, an antibody that specifically binds to human OX40 is an antibody, wherein the binding between the antibody and the variant OX40 is greatly weakened relative to the binding between the antibody and the human OX40 sequence of SEQ ID NO: 55 And wherein the variant OX40 includes the sequence of SEQ ID NO: 55, in addition to amino acid mutations selected from the group consisting of: N60A, R62A, R80A, L88A, P93A, P99A, P115A, And its combination.

In one embodiment, an antibody that specifically binds to human OX40 is an antibody, wherein the binding between the antibody and the variant OX40 is greatly weakened relative to the binding between the antibody and the human OX40 sequence of SEQ ID NO: 55 And wherein the variant OX40 includes the sequence of SEQ ID NO: 55, except for the following amino acid mutations: W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A.

In one embodiment, an antibody that specifically binds to human OX40 is an antibody that, compared with the human OX40 sequence that binds to SEQ ID NO: 55, displays the same protein as SEQ ID NO: 55 (except for the presence of A group of amino acid mutations, the group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof) reduced binding or non-binding.

In one embodiment, the antibody specifically binds to human OX40. An isolated antibody that specifically binds to human OX40. Compared with the human OX40 sequence that binds to SEQ ID NO: 55, the antibody displays the same protein as SEQ ID NO: 55 (except for the presence of amino acid mutations W58A, N60A, R62A). , R80A, L88A, P93A, P99A, and P115A) reduced binding or non-binding.

In one embodiment, an antibody that specifically binds to human OX40 is an antibody that specifically binds to an epitope of the human OX40 sequence, the epitope includes residues selected from the group consisting of SEQ ID NO: 55, and the group consists of the following Composition: 60, 62, 80, 88, 93, 99, 115, and combinations thereof.

In one embodiment, an antibody that specifically binds to human OX40 is an antibody that specifically binds to an epitope of the human OX40 sequence, and the epitope includes residues 58, 60, 62, 80, 88, 93 of SEQ ID NO: 55, 99, and 115.

In one embodiment, the antibody system that specifically binds to human OX40 specifically binds to an antibody of at least one residue selected from the group consisting of SEQ ID NO: 55, the group consisting of: 60, 62, 80, 88 , 93, 99, 115, and combinations thereof.

In one embodiment, the antibody includes a heavy chain variable region sequence and a light chain variable region sequence. The antibodies provided herein are selected from the group consisting of Fab, Fab', F(ab')2, And the scFv fragment.

In one embodiment, the antibody includes a human immunoglobulin IgG ₁ heavy chain constant region, and wherein _{the amino acid sequence of the IgG 1} heavy chain constant region includes a mutation selected from the group consisting of: N297A, N297Q, D265A, and their combinations. In one embodiment, the antibody includes an IgG ₁ heavy chain constant region, wherein _{the amino acid sequence of the IgG 1} heavy chain constant region includes a mutation selected from the group consisting of: D265A, P329A, and combinations thereof .

In one embodiment, an antibody that specifically binds to human OX40 includes: (a) a first antigen-binding domain that specifically binds to human OX40; and (b) a second antigen that does not specifically bind to an antigen expressed by human immune cells Combine domain.

In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: (a) the first heavy chain variable domain (VH) including the VH complementarity determining region (CDR) 1, and the VH CDR1 includes GSAMH (SEQ ID NO : 4) amino acid sequence; VH-CDR2, which includes the amino acid sequence of RIRSKANSYATAYAASVKG (SEQ ID NO: 5); and VH-CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); And (b) the first light chain variable domain (VL) including VL-CDR1, the VL-CDR1 including the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO:1); VL-CDR2, which includes LGSNRAS (SEQ ID NO : 2) the amino acid sequence; and VL- CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3). In one embodiment, the antigen-binding domain that specifically binds to human OX40 specifically binds to the same epitope of human OX40 as the following antibody, the antibody including: VH, which includes the amino acid sequence of SEQ ID NO: 16, and VL, which includes the amino acid sequence of SEQ ID NO:15. In one embodiment, compared to the human OX40 sequence that binds to SEQ ID NO: 55, the antigen-binding domain that specifically binds to human OX40 displays the same protein as SEQ ID NO: 55 (except for the presence of an amino group selected from the group below). Acid mutations, this group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof) reduced binding or non-binding. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes VH and VL, wherein VH includes the amino acid sequence of SEQ ID NO:16. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes VH and VL, where VL includes the amino acid sequence of SEQ ID NO:15.

In one embodiment, the second antigen binding domain specifically binds to a non-human antigen. In one embodiment, the second antigen binding domain specifically binds viral antigens. In one embodiment, the viral antigen is an HIV antigen. In one embodiment, the second antigen binding domain specifically binds chicken albumin or chicken egg lysozyme.

In one embodiment, the antibody that specifically binds to human OX40 includes: (a) the first heavy chain variable domain (VH) including the VH complementarity determining region (CDR) 1, and the VH CDR1 includes GSAMH (SEQ ID NO: 4 ) Of the amino acid sequence; VH-CDR2, which includes The amino acid sequence of RIRSKANSYATAYAASVKG (SEQ ID NO: 5); and VH-CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6); and (b) the first light chain variable including VL-CDR1 Domain (VL), the VL-CDR1 includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 1); VL-CDR2, which includes the amino acid sequence of LGSNRAS (SEQ ID NO: 2); and VL-CDR3, which Including the amino acid sequence of MQALQTPLT (SEQ ID NO: 3). In one embodiment, the antigen-binding domain that specifically binds to human OX40 specifically binds to the same epitope of human OX40 as the following antibody, the antibody including: VH, which includes the amino acid sequence of SEQ ID NO: 16, and VL, which includes the amino acid sequence of SEQ ID NO:15. In one embodiment, compared to the human OX40 sequence that binds to SEQ ID NO: 55, the antigen-binding domain that specifically binds to human OX40 displays the same protein as SEQ ID NO: 55 (except for the presence of an amino group selected from the group below). Acid mutations, this group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof) reduced binding or non-binding. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes VH and VL, wherein VH includes the amino acid sequence of SEQ ID NO:16. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes VH and VL, where VL includes the amino acid sequence of SEQ ID NO:15.

In one embodiment, the second heavy chain is an Fc fragment.

In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VH, which includes an amino acid with SEQ ID NO: 16 An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical in sequence. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VH, which includes the amino acid sequence of SEQ ID NO:16. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VH, which includes an amino acid sequence derived from the germline sequence of human IGHV3-73.

In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VL, which includes at least 75%, 80%, 85%, 90%, 95%, or 99% of the amino acid sequence of SEQ ID NO: 15 % Consistent amino acid sequence. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VL-CDR3, which includes the amino acid sequence of SEQ ID NO:3. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VL, which includes the amino acid sequence of SEQ ID NO:15. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes a light chain, which includes the amino acid sequence of SEQ ID NO:20. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: a light chain, which includes the amino acid sequence of SEQ ID NO:50. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: VL, which includes an amino acid sequence derived from the germline sequence of human IGKV2-28.

In one embodiment, the antigen binding domain that specifically binds to human OX40 includes the VH and VL sequences listed in SEQ ID NOs: 16 and 15, respectively. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes: a heavy chain, which includes the amine group of SEQ ID NO: 21 Acid sequence. In one embodiment, the antigen binding domain that specifically binds to human OX40 includes a heavy chain, which includes the amino acid sequence of SEQ ID NO:60.

In one embodiment, the first antigen binding domain and the second antigen binding domain include the same mutation selected from the group consisting of N297A, N297Q, D265A, and combinations thereof. In one embodiment, the first antigen-binding domain and the second antigen-binding domain include the same mutation selected from the group consisting of D265A, P329A, and combinations thereof. In one embodiment, the antigen-binding domain that specifically binds to human OX40 and the second heavy chain or fragments thereof includes the same mutation selected from the group consisting of: N297A, N297Q, D265A, and combinations thereof . In one embodiment, the antigen binding domain that specifically binds to human OX40 and the second heavy chain or fragments thereof includes the same mutation selected from the group consisting of D265A, P329A, and combinations thereof.

In one embodiment, the antibody is antagonistic to human OX40. In one embodiment, the antibody inactivates, reduces or inhibits the activity of human OX40. In one embodiment, the antibody inhibits or reduces the binding of human OX40 to the human OX40 ligand. In one embodiment, the antibody inhibits or reduces human OX40 signaling. In one embodiment, the antibody inhibits or reduces human OX40 signaling induced by the human OX40 ligand.

In one embodiment, the antibody includes a detectable label.

In one embodiment, the invention relates to the use of an antibody of the invention as a medicament.

In one embodiment, the present invention relates to the use of the antibody of the present invention as a diagnostic agent.

In one embodiment, the present invention relates to the use of the antibody of the present invention to detect OX40 in a sample in vitro. In one embodiment, OX40 is human OX40.

In one embodiment, the present invention relates to the use of the antibody of the present invention for initiating, enhancing or inducing the activity of human OX40 in vitro. In one embodiment, the antibody induces CD4+ T cell proliferation in vitro.

In one aspect, provided herein is an isolated nucleic acid molecule encoding an antibody that specifically binds to OX40 (e.g., human OX40). In one embodiment, the nucleic acid molecule encodes the heavy chain variable region or heavy chain of the anti-OX40 antibody provided herein. In one embodiment, the nucleic acid molecule encodes the light chain variable region or light chain of an anti-OX40 antibody provided herein. In one embodiment, the nucleic acid molecule encodes the heavy chain variable region or heavy chain of the anti-OX40 antibody provided herein and the light chain variable region or light chain of the antibody. In one embodiment, the nucleic acid molecule encodes a heavy chain variable region, which includes the amino acid sequence of SEQ ID NO:16. In one embodiment, the nucleic acid molecule encodes a light chain variable region, which includes the amino acid sequence of SEQ ID NO:15. Also provided here are isolated antibodies encoded by such nucleic acid molecules.

In one aspect, provided herein are vectors containing such nucleic acid molecules.

In one aspect, provided herein is a host cell comprising such nucleic acid molecule or such vector. In one embodiment, the host cell is selected from the group consisting of: Escherichia coli in tissue culture, Pseudomonas Sporus, Bacillus, Streptomyces, Yeast, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells and human cells.

In one aspect, provided herein is a method of producing antibodies that specifically bind to OX40 (e.g., human OX40), the method comprising culturing such host cells so that nucleic acid molecules are expressed and antibodies are produced.

In one embodiment, the present invention relates to the use of the antibody of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, and/or the host cell of the present invention as a medicine.

In one embodiment, the present invention relates to the use of the antibody of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, and/or the host cell of the present invention as a diagnostic agent.

In one embodiment, the present invention relates to the use of the antibody of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, and/or the host cell of the present invention for detecting OX40 in a sample in vitro. In one embodiment, OX40 is human OX40.

In one embodiment, the present invention relates to the use of the antibody of the present invention for initiating, enhancing or inducing the activity of human OX40 in vitro. In one embodiment, the antibody induces CD4+ T cell proliferation in vitro.

In one aspect, a pharmaceutical composition is provided, the pharmaceutical composition includes the antibody that specifically binds to OX40 provided herein, encoding A nucleic acid molecule of an antibody that specifically binds to OX40 (for example, human OX40), a vector containing such a nucleic acid molecule, or a host cell containing such a nucleic acid molecule or vector.

In one aspect, there is provided a pharmaceutical composition, the pharmaceutical composition comprising an antibody that specifically binds to OX40 provided herein, a nucleic acid molecule encoding an antibody that specifically binds to OX40 (for example, human OX40), and a nucleic acid molecule comprising such a nucleic acid molecule The vector, or the host cell containing such nucleic acid molecule or vector, is used as a medicine.

In one aspect, there is provided a pharmaceutical composition, the pharmaceutical composition comprising an antibody that specifically binds to OX40 provided herein, a nucleic acid molecule encoding an antibody that specifically binds to OX40 (for example, human OX40), and a nucleic acid molecule comprising such a nucleic acid molecule The vector, or the host cell containing such nucleic acid molecule or vector, is used as a diagnostic agent.

In one aspect, provided herein is a method for modulating an immune response of a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, host cell, or pharmaceutical composition provided herein. In one embodiment, the method is used to enhance or induce an immune response in a subject. In one embodiment, modulating the immune response includes enhancing or inducing the immune response of the subject.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of modulating an immune response.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in enhancing or inducing Used in methods to induce immune response. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of modulating the immune response of a subject.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of enhancing or inducing an immune response in a subject. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of modulating the immune response of a subject, the method comprising administering to the subject an effective An amount of the antibody, nucleic acid, vector, host cell, or pharmaceutical composition of the present invention.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of enhancing or inducing an immune response in a subject, the method comprising giving the subject An effective amount of the antibody, nucleic acid, vector, host cell, or pharmaceutical composition of the present invention is administered. In one embodiment, the resistance system is agonistic.

In one aspect, there is provided a method for enhancing T cell expansion and T cell effector function in a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, Host cell, or pharmaceutical composition.

In one aspect, the present invention relates to the antibody, nuclear Acids, vectors, host cells, and/or pharmaceutical compositions are used in methods for enhancing T cell expansion and T cell effector functions. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method for enhancing T cell expansion and T cell effector function in a subject. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method for enhancing T cell expansion and T cell effector function in a subject, the method This includes administering to the subject an effective amount of the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention. In one embodiment, the resistance system is agonistic.

In one aspect, provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, host cell, or pharmaceutical composition provided herein. In some embodiments, the cancer is selected from the group consisting of melanoma, kidney cancer, and prostate cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, colon cancer, and lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In one example, the method further includes administering a checkpoint targeting agent to the subject. In one example, the checkpoint targeting agent is selected from the group consisting of: antagonist anti-PD-1 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-CTLA-4 anti- Antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-GITR antibody, agonist anti-CD137 antibody, and agonist anti-OX40 antibody.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of The antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) checkpoint targeting agent for use as a medicine. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) checkpoint targeting agent for use in a method of treating cancer. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to pharmaceutical compositions, kits Or a multi-part kit, which includes (a) the antibody, nucleic acid, vector, host cell, and/or medical composition of the present invention and (b) a checkpoint targeting agent.

The antibodies as described herein can be used in combination with IDO inhibitors to treat cancer. In one embodiment, the method further comprises administering to the subject an inhibitor of indoleamine-2,3-dioxygenase (IDO). The IDO inhibitors used for the treatment of cancer as described herein exist in solid dosage forms (such as tablets, pills, or capsules) of a pharmaceutical composition, wherein the pharmaceutical composition includes the IDO inhibitor and a pharmaceutically acceptable excipient. Therefore, the antibody as described herein and the IDO inhibitor as described herein can be administered separately, sequentially or simultaneously as separate dosage forms. In one embodiment, the antibody is administered parenterally, while the IDO inhibitor is administered orally. In a specific embodiment, the inhibitor is selected from the group consisting of: epacadostat (Incyte Corporation), F001287 (Flexus Biosciences), Indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). Epacadostat has been described in PCT Publication No. WO 2010/005958, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the inhibitor is epacadostat. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indoximod. In another embodiment, the inhibitor is NLG919.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) IDO inhibitor for use as a medicine.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) IDO inhibitor for use in a method of treating cancer. In one aspect, the present invention relates to a composition, kit or multi-part kit comprising (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the invention and (b) an IDO inhibitor.

The antibodies described here can be used in combination with vaccines. In a specific embodiment, the vaccine includes a heat shock protein peptide complex (HSPPC), wherein the HSPPC includes a heat shock protein (e.g., gp96 protein, hsp70 protein, Or hsc70 protein). In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigen peptide. In one embodiment, the heat shock protein is hsp70 or hsc70 protein and is complexed with a tumor-associated antigen peptide. In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigen peptide, wherein the HSPPC is derived from a tumor obtained from the subject. In one embodiment, the heat shock protein is hsp70 or hsc70 protein and is complexed with a tumor-associated antigen peptide, wherein the HSPPC is derived from a tumor obtained from the subject.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) vaccine for use as a medicine. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention and (b) vaccine for use in a method of treating cancer. In one embodiment, the resistance system is agonistic.

In one aspect, the present invention relates to a composition, kit or multi-part kit, which includes (a) the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the invention and (b) a vaccine. In one embodiment, the resistance system is agonistic.

In some embodiments, the present disclosure provides the use of antibodies as described herein in the manufacture of drugs for the treatment of cancer. In certain embodiments, the present disclosure provides antibodies as described herein for use in the treatment of cancer. In certain embodiments, the present disclosure provides the use of the pharmaceutical composition as described herein in the manufacture of drugs for the treatment of cancer. In certain embodiments, the present disclosure provides a pharmaceutical composition as described herein for use in the treatment of cancer.

In one aspect, provided herein is a method for treating autoimmune or inflammatory diseases or disorders in a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, host cell, Or pharmaceutical composition. In some embodiments, the disease or disorder is selected from the group consisting of transplant rejection, vasculitis, asthma, rheumatoid arthritis, dermatitis, inflammatory bowel disease, uveitis, and lupus.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating autoimmune or inflammatory diseases or disorders. In one embodiment In, anti-system antagonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating autoimmune or inflammatory diseases or disorders in a subject. In one embodiment, the resistance system is antagonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method for treating an autoimmune or inflammatory disease or disorder in a subject, the method comprising: The subject is administered an effective amount of the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention. In one embodiment, the resistance system is antagonistic.

In one aspect, there is provided a method for treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, host cell, or pharmaceutical composition provided herein .

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating infectious diseases. In one embodiment, the resistance system is agonistic. In one embodiment, the resistance system is antagonistic.

In one aspect, the present invention relates to the antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition of the present invention for use in a method of treating an infectious disease in a subject. In one embodiment, the resistance system is agonistic. In one embodiment, the resistance system is antagonistic.

In one aspect, the present invention relates to the antibody, nuclear The acid, vector, host cell, and/or pharmaceutical composition are used in a method for treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody, nucleic acid, vector, Host cells, and/or pharmaceutical compositions. In one embodiment, the resistance system is agonistic. In one embodiment, the resistance system is antagonistic.

In one embodiment of the methods provided herein, the subject is a human.

In one aspect, provided herein is a method for detecting OX40 in a sample, the method comprising contacting the sample with the antibody provided herein.

In one aspect, provided herein is a method for detecting OX40 in a sample in vitro, the method comprising contacting the sample with the antibody provided herein. In one embodiment, OX40 is human OX40.

In one aspect, provided herein is a method for detecting OX40 in a sample in vitro, the method comprising contacting the sample with an antibody, nucleic acid, vector, host cell, and/or pharmaceutical composition provided herein. In one embodiment, OX40 is human OX40.

In one aspect, the antibodies, nucleic acids, vectors, host cells, and/or pharmaceutical compositions provided herein are provided herein. Preferably, the antibodies provided herein are used to detect OX40 in a sample in vitro.

In one aspect, the antibodies, nucleic acids, vectors, host cells, and/or pharmaceutical compositions provided herein are provided herein, preferably the antibodies provided herein are for use in detecting OX40 in a subject. In one embodiment, the subject is human.

In one aspect, a kit is provided, the kit includes: the antibodies provided herein that specifically bind to OX40, nucleic acid molecules encoding antibodies that specifically bind to OX40 (for example, human OX40), and nucleic acid molecules comprising such nucleic acid molecules A vector, a host cell containing such a nucleic acid molecule or vector, or a pharmaceutical composition containing such an antibody, nucleic acid molecule, vector, or host cell, and a) detection reagent, b) OX40 antigen, and c) identification, the identification reflects Approved for human administration, use or sale, or d) a combination thereof.

Figures 1A, 1B, 1C, 1D, and 1E: Figure 1A is a pair of bar graphs showing the binding of anti-OX40 antibody pab1949 and isotype control to activated human CD4+ T cells and CD8+ T cells. Figure 1B is a pair of bar graphs showing the binding of pab1949-1 and isotype control to unstimulated and stimulated (using anti-CD3 antibody) CD4+ T cells. Figure 1C is a graph showing the binding of dose-titrated pab1949-1 or isotype control to activated human CD4+ T cells. Figure 1D is a set of bar graphs measuring the binding of pab1949-1 and isotype controls to human unstimulated blood-derived immune cell populations. Fig. 1E is a bar graph showing the binding of pab1949 and isotype control to activated cynomolgus monkey ( Macaca fascicularis ) CD4+ T cells.

Figures 2A, 2B, and 2C are graphs showing the results of suboptimal CD3 stimulation assays to evaluate the effects of stimulation of anti-OX40 antibodies pab1949 (Figures 2A and 2C) and pab2044 (Figure 2B) on the proliferation of enriched CD4+ T cells. The antibody pab1949 is a human IgG ₁ antibody. Antibodies pab2044 and pab1949 have the same heavy chain variable region and the same light chain, but include the human IgG ₄ constant region. The cell proliferation (CFSE; x-axis) is shown for each antibody tested: IgG ₁ isotype control, pab1949, and anti-CD28 antibody in _{Figure 2A as positive controls; and IgG 4} isotype control, pab2044, and Figure 2B Anti-CD28 antibody. Figure 2C is a line graph showing the titration of the anti-OX40 antibody pab1949 (0.002, 0.02, 0.2, 2, and 20 μg/ml) under suboptimal anti-CD3 stimulation and the effect of this antibody on the proliferation of enriched CD4+ T cells.

Figure 3A, 3B, 3C, 3D, 3E, and 3F are representative of the production of IFNγ and TNFα cytokines induced by the combination of anti-OX40 antibody pab1949 or pab1949-1 with different suboptimal concentrations of anti-CD3 antibody and IL-2 The results of the sexual analysis. In Figures 3A-3C, PBMCs from four different donors were tested: Donor KM, Donor TM, Donor GS, and Donor SB. Figures 3A and 3B show plots of intracellular interleukin staining (IFNγ and TNFα) of CD4+ T cells and CD8+ T cells from donor SB (Figure 3A) and donor GS (Figure 3B). For the anti-OX40 antibody pab1949 or isotype control, the percentages of IFNγ + TNFα + multifunctional CD4 + T cells and CD8 + T cells or TNFα + monofunctional CD4 + T cells and CD8 + T cells are plotted (Figure 3C). The percentages shown in Figure 3C represent the highest response produced at three different anti-CD3 antibody concentrations. Error bars represent standard deviation (n=2). Figures 3D, 3E, and 3F are a set of graphs showing the dose titration of _{anti-OX40 antibody pab1949-1 or IgG 1} isotype control antibody in cells derived from donor GS-derived PBMC in a similar suboptimal anti-CD3 stimulation assay The percentages of induced TNFα+CD4+ T cells (Figure 3D), IFNγ+TNFα+multifunctional CD8+ T cells (Figure 3E), and IFNγ+CD8+ T cells (Figure 3F).

Figures 4A, 4B, and 4C are a set of graphs showing the results of suboptimal anti-CD3 stimulation assays using cells derived from donors 1, 2, 4, 5, 7, 8, 9, and 10. The antibody concentration range against pab1949-1 or IgG ₁ isotype control antibody is plotted against the percentage of IFNγ+, TNFα+, or IFNγ+ TNFα+ multifunctional CD4+ or CD8+ T cells.

Figure 5A is a set of horizontal bar graphs, showing the use of PBMC from donor SB and donor GS, anti-OX40 antibody pab1949 or isotype control to a series of cytokines (IL-2, TNFα) in the suboptimal anti-CD3 stimulation assay. , IL-10, IL-4, and IL-13) secretion. Use different suboptimal concentrations of anti-CD3 antibody (strain SP34), IL2 (20U/ml), and 5 μg/ml anti-OX40 antibody or IgG ₁ isotype control to activate PBMC from healthy donors, and 4 days after stimulation (SB#1A) or 3 days (SB#1B, SB#2, and GS) to measure cytokines. The bars in Figure 5A represent the highest cytokine secretion induced by all anti-CD3 concentrations tested at an anti-OX40 antibody concentration of 5 μg/ml. Error bars represent standard deviation (n=2). Figures 5B, 5C, and 5D are a set of graphs showing that in the presence of suboptimal concentrations of anti-CD3 antibodies, different concentrations of pab1949-1 or IgG ₁ isotype control antibodies are induced in cells derived from donor GS-derived PBMC The secretion of cytokines (TNFα, IL-10, or IL-13).

Figures 6A, 6B, and 6C are _{series of graphs showing the GM induced by pab1949-1 or IgG 1} isotype control antibody dose titration in the suboptimal anti-CD3 stimulation assay in cells derived from donor 1-10 PBMC -The amount of CSF secreted. ECL refers to electrochemiluminescence.

FIG 7A, 7B, and 7C is similar to FIG 6A, 6B, and 6C, showing secretion of pab1949-1 or IgG ₁ isotype control antibody induced a dose titration of IL-2. ECL refers to electrochemiluminescence.

FIGS. 8A, 8B, and 8C is similar to Figure 6A, 6B, and 6C, it shows TNFβ secretion by pab1949-1 or IgG ₁ isotype control antibody induced dose titration.

Figures 9A and 9B are bar graphs showing that regulatory T cells (Treg) and effector T cells (Teff) (Treg: Teff) co-cultured at a ratio of 1:3 are composed of soluble or cross-linked (using Anti-FcF(ab') ₂ )pab1949-1 or IgG ₁ isotype control antibody induced IL-2 (Figure 9A) and IL-10 (Figure 9B) production.

Figures 10A, 10B, 10C, 10D, 10E, 10F, and 10G are graphs depicting the functional activity of anti-OX40 antibodies on primary human PBMCs upon staphylococcal enterotoxin A (SEA) stimulation. In the absence or presence of a fixed concentration (10 μg/ml) or different concentrations of anti-OX40 antibodies or isotype controls, human PBMCs were stimulated with SEA and the secretion of IL-2 or IL-10 cytokines was assessed. The anti-OX40 antibodies tested included pab1949, pab1949-1, pab2193-1, pab1949-1-N297A, and reference antibodies pab1784 and pab2045. At an anti-OX40 antibody concentration of 10 μg/ml, the fold change of the tested antibodies to IL-2 (Figure 10A) and IL-10 (Figure 10B) was plotted. Figures 10C, 10D, and 10E show the dose response curves of the fold change of IL-2 at different concentrations of pab1949, pab1949-1, or reference antibodies pab1784 and pab2045. The statistical significance is determined by the Student's t-test compared to the isotype control sample indicated by the asterisk. Error bars represent the standard deviation from 3 replicates. * In Figure 10A represents p<0.001. * In Figure 10B represents p<0.01. Figure 10F is a series diagram showing IL-2 production induced by dose titration of _{pab1949-1, pab2193-1, IgG 1} isotype control antibody, or IgG _{2 isotype control antibody.} Figure 10G is a series diagram showing IL-2 production in response to dose titration of pab1949-1, pab1949-1-N297A, or IgG _{1 isotype control antibody.}

Figures 11A and 11B are the results from the assay in which the OX40 NF-κB-luciferase reporter (reporter) cell line is soluble (soluble conditions, Figure 11A) or cross-linked (complex conditions, figure 11B) Pab1949-1 or IgG ₁ isotype control antibody for testing. Relative light units (RLU) are plotted against the different antibody concentrations tested.

Figures 12A and 12B are the results from the reporter assay in which it was tested that the anti-OX40 antibody was used to activate the expression FcγRIIIA (Figure 12A) or the FcγRIIAH131 variant (Figure 12B) when the anti-OX40 antibody binds to target cells expressing OX40. ) The ability of the reporter cell. In Figure 12A, the ΔRLU values are plotted for different concentrations of pab1949-1 and pab2044-1. △RLU represents the RLU of the anti-OX40 antibody minus the RLU of the isotype control. In Figure 12B, RLU values are plotted against increasing concentrations of pab1949-1, pab1949-1-S267E/L328F, pab2193-1, IgG ₁ isotype control antibody or IgG _{2 isotype control antibody.}

Figure 13A is a bar graph showing the ΔMFI of human OX40 on nTregs, CD4+ T cells, or CD8+ T cells from healthy donors activated by anti-CD3/anti-CD28 beads (as measured by flow cytometry) measured). △MFI represents the MFI of the anti-OX40 antibody minus the MFI of the isotype control. The anti-OX40 antibody system used was PE-conjugated mouse anti-human OX40 antibody (Biolegend: ACT35; catalog: 350004; batch number: B181090). Figure 13B shows a bar graph of the ΔMFI of human OX40 on activated nTreg from two healthy donors and human OX40 on effector T cells. The cells were stained with commercial anti-OX40 antibody (BER-ACT35 clone) or isotype control antibody and analyzed using flow cytometry. Figure 13C is a diagram showing the use of an Fcγ receptor IIIA (FcγRIIIA) reporter cell line to test the anti-OX40 antibody pab1949. At 37°C, in the presence of pab1949 or isotype control, Jurkat NFAT-luciferase reporter cells overexpressing FcγRIIIA (158 V/V polymorphism) were co-cultured with activated primary nTreg and effector T cells for continuous co-culture 20 hours. After 20 hours, the relative light unit (RLU) is recorded, which represents FcyRIIIA binding. △RLU represents the RLU of the anti-OX40 antibody minus the RLU of the isotype control. Error bars represent standard deviation (n=2). The data shown are representative of experiments using cells from three donors. Figure 13D is similar to Figure 13C and shows results from a study that tested pab1949-1 using a modified protocol.

Figure 14A is a set of bar graphs showing the surface performance of OX40 measured by flow cytometry. Samples were collected from the blood of healthy human donors (ac, n=3) or tumor tissues from non-small cell lung cancer patients (NSCLC) (df, n=3). The cell population is defined as follows: Tconv (CD3+, CD4+, CD8a-, CD25 low, and FOXP3-) or Treg (CD3+, CD4+, CD8a-, CD25 high, and FOXP3+). Figure 14B is a pair of bar graphs from a study similar to that depicted in Figure 14A, measuring the surface OX40 expression in CD8+ or CD4+ T cells, or Treg cells from endometrial cancer samples. Figure 14C line shows a bar graph showing OX40 expression on Treg cells and Teff cells in different tumor types. Figure 14D is a table summarizing the expression of OX40 in tumor-related CD4+ Teff cells and Treg cells. "-" represents negative/no performance, "+" represents weak performance, "++" represents moderate performance, and "+++" represents high performance.

15A and 15B based a set of graphs display the amount of antibody secretion induced a dose titration of GM-CSF in cynomolgus monkey PBMC derived from cells or control by a pab1949-1 isotype IgG _1. Note that Cyno 2 and Cyno 9 refer to PBMCs from the same cynomolgus monkey tested in independent experiments. All other PBMC samples were collected from different cynomolgus monkey donors.

FIGS 16A and 16B is similar to FIGS. 15A and 15B, shows the IL-17 secretion by pab1949-1 or IgG ₁ isotype control antibody induced dose titration.

17A and 17B is similar to FIGS. 15A and 15B, shows TNFβ secretion by pab1949-1 or IgG ₁ isotype control antibody induced dose titration.

Figures 18A and 18B are similar to Figures 15A and 15B and show the secretion of IL-5 induced by dose titration of _{pab1949-1 or IgG 1 isotype control antibody.}

Figures 19A and 19B are similar to Figures 15A and 15B and show the secretion of IL-10 induced by dose titration of _{pab1949-1 or IgG 1 isotype control antibody.}

Figures 20A and 20B are a set of graphs showing the results of testing the functional activity of anti-OX40 antibody pab1949-1 on primary cynomolgus monkey PBMC when stimulated by staphylococcal enterotoxin A (SEA). The amount of IL-2 secreted by PBMC from two cynomolgus monkey donors was plotted against the dose titration of pab1949-1 or IgG _{1 isotype control antibody.}

Figure 21 is a table summarizing the binding of monoclonal anti-OX40 antibodies pab1949-1 and pab1928 to 1624-5 cells expressing human OX40 alanine mutants.

Provided herein is an antibody (e.g., a monoclonal antibody) that specifically binds to OX40 (e.g., human OX40) and modulates the activity of OX40. For example, in one aspect, provided herein are antibodies that specifically bind to OX40 and enhance, induce, or increase one or more OX40 activities. For example, in another aspect, there is provided herein that specifically binds to OX40 (e.g., human OX40) and inactivates, reduces or inhibits one or more OX40 activities Of antibodies. In a specific embodiment, the anti-system is isolated.

Also provided are isolated nucleic acids (polynucleotides) encoding such antibodies, such as complementary DNA (cDNA). Further provided are vectors (e.g., expression vectors) and cells (e.g., host cells), which include nucleic acids (polynucleotides) encoding such antibodies. Methods of making such antibodies are also provided. In other aspects, methods and uses for inducing, increasing or enhancing OX40 activity and treating certain diseases (such as cancer) are provided herein. Further provided are methods and uses for inactivating, reducing, or inhibiting the activity of OX40 (for example, human OX40) and treating certain diseases (such as inflammatory or autoimmune diseases and disorders). Related components (for example, pharmaceutical components), kits, and detection methods are also provided.

5.1 Terminology

As used herein, the terms "about" and "approximately", when used to modify a value or a range of values, indicate that the deviation of 5% to 10% above and 5% to 10% below the value or range is maintained at The value or range is intended to be within the intended range.

As used here, if within a specified domain (for example, in a given experiment, or using the average of multiple experiments) B increases substantially as A increases, then within the specified domain of the value of A, B is the "basically increasing function" of A. This definition allows the B value corresponding to a given value of A to be 1%, 2%, 3%, 4%, 5%, 10%, 15%, or lower than the B value corresponding to any lower value of A 20%.

As used herein, the term "antibody" is a term used in the art And it can be used interchangeably here, and means a molecule having an antigen binding site that specifically binds to an antigen.

Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies, including Two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, Heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFv), camelized antibodies, affinities (affybody), Fab fragments, F(ab') ₂ fragments, Disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. In certain embodiments, the anti-system described herein refers to a multi-strain antibody population. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any type (e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , or IgA ₂ ) or any subclass ( For example, IgG _2a or IgG _2b ) immunoglobulin molecules. In certain embodiments, the antisystemic IgG antibody described herein, or its class (e.g., human IgG ₁ , IgG ₂ , or IgG ₄ ) or subclass. In a specific embodiment, the anti-system humanized monoclonal antibody. In another specific embodiment, the anti-systemic human monoclonal antibody is, for example, an immunoglobulin. In certain embodiments, the antibodies described herein are IgG ₁ , IgG ₂ , or IgG ₄ antibodies.

As used herein, the terms "antigen binding domain", "antigen "Binding region", "antigen binding site" and similar terms refer to the portion of an antibody molecule that includes amino acid residues (e.g., complementarity determining region (CDR)) that confer specificity to the antibody molecule against an antigen. Antigen binding region It can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans.

As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically refers to the part of the antibody, generally speaking, the part of the light or heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino end of the mature heavy chain And about 90 to 115 amino acids in the mature light chain, which differ widely in sequence between antibodies and are used for the binding and specificity of specific antibodies for their specific antigens. Sequence variability is concentrated in those regions called complementarity determining regions (CDR), while the more highly conserved regions in variable domains are called framework regions (FR). Without wishing to be bound by any specific mechanism or theory, it is believed that the CDRs of the light chain and the heavy chain are mainly responsible for the interaction and specificity between the antibody and the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable regions include rodent or murine CDRs and human framework regions (FR). In a specific embodiment, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable regions include rodent or murine CDRs and primate (e.g., non-human primates) framework regions (FR).

The terms "VL" and "VL domain" are used interchangeably and refer to the variable region of the light chain of an antibody.

The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of the heavy chain of an antibody.

The term "Kabat numbering" and similar terms are recognized in the art and refer to the system of numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen binding portions. In some aspects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat (Kabat) EA & Wu (Wu) TT (1971) Ann NY Acad Sci) 190:382- 391 and Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH publication number 91-3242). Using the Kabat numbering system, the CDRs in the antibody heavy chain molecule typically exist in amino acid positions 31 to 35, which can optionally include one or two additional amino acids after 35 (in the Kabat numbering scheme) Referred to as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule typically exist in amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

As used herein, the terms "constant region" or "constant domain" are interchangeable and have a common meaning in the art. The constant region is the part of the antibody, for example, the carboxy terminal part of the light chain and/or heavy chain, which does not directly participate in It binds to antibodies and antigens, but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have more conservative amino acid sequences than immunoglobulin variable domains.

As used herein, when used in relation to antibodies, the term "heavy chain", the amino acid sequence based on the constant domain can mean any of the different types, for example, α, δ, ε, γ, and μ, which respectively produce antibodies The types of IgA, IgD, IgE, IgG, and IgM include subclasses of IgG, for example, IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .

As used herein, when used in relation to antibodies, the term "light chain", the amino acid sequence based on the constant domain can mean any of different types, for example, kappa (κ) or lambda (λ). The light chain amino acid sequence is well known in the art. In a specific embodiment, the light chain is a human light chain.

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X to its partner Y can usually be represented by the dissociation constant (K _D ). Affinity can be measured and/or expressed in a variety of ways known in the art, including, but not limited to, equilibrium dissociation constant (K _D ) and equilibrium association constant (K _A ). _{K D is calculated from} the quotient of k _off /k on, and _{K A is} calculated from the quotient of _{k on} /k _off . k means the _opening rate, for example, an antigen binding antibody constant, k _off and, for example, refers to an antibody from the antigen solution. _{The k-on} and k- _off can be determined by a technique known to those who are familiar with the technology, such as BIAcore ^® or KinExA.

As used herein, "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. The family of amino acid residues with side chains has been defined in the art. These families include amino acids with the following: basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), without an electrical side Chain (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine) Acid, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (such as threonine, valine, isoleucine) and aromatic side Chain (e.g. tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in one or more CDRs or in one or more framework regions of an antibody may be substituted with amino acid residues having similar side chains.

As used herein, "epitope" is a term in the art and refers to a local area of an antigen to which an antibody can specifically bind. The epitope can be, for example, the continuous amino acid of the polypeptide (linear or continuous epitope) or the epitope can be, for example, two or more non-contiguous regions from one or more polypeptides gathered together (conformation, nonlinear , Discontinuous or discontinuous epitopes). In some embodiments, the epitope bound by the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA analysis, coupled mass spectrometry (e.g., liquid chromatography mass spectrometry) hydrogen/deuterium exchange, based on The oligopeptide scanning analysis of the array, and/or mutagenic gene mapping (for example, site-directed mutagenesis gene mapping) is determined. For X-ray crystallography, any method known in the art can be used to complete crystallization (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1 -23; Chayen NE (1997) Structure 5: 1269-1274; Macpherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be studied using the well-known X-ray diffraction technique and computer software (such as X-PLOR (Yale University), 1992, refined by Molecular Simulations, Inc.); See, for example, Meth Enzymol (1985) Volumes 114 and 115, edited by Wyckoff HW et al.; US2004/0014194), and BUSTER (Bricogne G (1993) ) Acta Crystallography Series D: Biocrystallography 49 (Pt 1): 37-60; Brikonai G (1997) Methods of Enzymology 276A: 361-423, edited by Carter CW; Roversi P Et al., (2000) Acta Crystallography Series D: Biological Crystallography 56 (Pt 10): 1316-1323). Mapping studies of mutagenic genes can be done using any method known to those skilled in the art. See, for example, Champe M et al. (1995) Journal of Biological Chemistry 270:1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085 on mutagenesis technology The description includes alanine partitioned mutagenesis technology. In a specific embodiment, alanine scanning mutagenesis studies are used to determine the epitope of the antibody.

As used herein, the terms "immune-specific binding", "immune-specific recognition", "specific binding" and "specific recognition" are similar terms in the context of antibodies and refer to binding to an antigen (e.g., table Or immune complexes), and this combination is understood by those familiar with the technology. For example, a molecule that specifically binds to an antigen usually binds to other peptides or polypeptides with lower affinity, which can be used, for example, by immunoassay, BIAcore ^® , KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho). (ID)) or other analysis known in the art. In one specific embodiment, immunospecifically binds to an antigen molecule at least 2 log, 2.5 log 3 log 4 log or greater than K _A time when molecules non-specifically bind to another antigen K _A incorporated into the antigen. In the context of an antibody having an anti-OX40 antigen-binding domain and a second antigen-binding domain that does not specifically bind to an antigen expressed by human immune cells, the terms "immune specific binding", "immune specific recognition", "specific binding" And "specific recognition" refers to antibodies that have outstanding specificity for more than one antigen (ie, OX40 and antigen related to the second antigen-binding domain).

In another specific embodiment, molecules that immunospecifically bind to antigens do not cross-react with other proteins under similar binding conditions. In another specific embodiment, molecules that immunospecifically bind to antigens do not cross-react with other non-OX40 proteins. In a specific embodiment, provided herein is an antibody that binds to OX40 with a higher affinity than another unrelated antigen. In some embodiments, it is provided here that 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95% affinity or higher affinity than another unrelated antigen binding to OX40 (for example, human OX40) Of antibodies, as measured by, for example, radioimmunoassay, plasmon resonance, or kinetic exclusion assays. In a specific embodiment, the degree of binding of an anti-OX40 antibody described herein to an unrelated non-OX40 protein is less than 10%, 15%, or 20% of the binding of the antibody to OX40 protein, as measured by, for example, radioimmunoassay .

In a specific embodiment, provided herein is an antibody that binds to human OX40 with a higher affinity than another OX40. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% are provided here. , 70% affinity or higher affinity than another OX40 antibody that binds to human OX40, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a specific embodiment, an antibody that binds to human OX40 described herein will bind to another OX40 protein by less than 10%, 15%, or 20% of the antibody's binding to human OX40 protein, such as by radioimmunoassay, surface Measured by plasmon resonance or kinetic exclusion assay.

As used herein, the term "OX40 receptor" or "OX40" or "OX40 polypeptide" refers to OX40 including, but not limited to, natural OX40, subtypes of OX40, or interspecies OX40 homologs of OX40. Ox40 is also known as Tumor Necrosis Factor Receptor Superfamily Member 4 (TNFRSF4), ACT35, CD134, IMD16, and TXGP1L. GenBank ^TM accession numbers BC105070 and BC105072 provide human OX40 nucleic acid sequences. Refseq number NP_003318.1 provides the amino acid sequence of human OX40. The immature amino acid sequence of human OX40 is provided as SEQ ID NO:17. The mature amino acid sequence of human OX40 is provided as SEQ ID NO:55. Human OX40 is designated by Entrez Gene as GeneID: 7293. RefSeq numbers XM_005545122.1 and XP_005545179.1 provide the predicted nucleic acid sequence and amino acid sequence of cynomolgus monkey OX40, respectively. A soluble subtype of human OX40 has also been reported (Taylor L et al. (2001) Journal of Immunological Methods 255: 67-72). As used herein, the term "human OX40" refers to OX40 including the polypeptide sequence of SEQ ID NO:55.

As used herein, the terms "OX40 ligand" and "OX40L" refer to tumor necrosis factor ligand superfamily member 4 (TNFSF4). OX40L is also known as CD252, GP34, TXGP1, and CD134L. GenBank ^TM accession numbers D90224.1 and AK297932.1 provide exemplary human OX40L nucleic acid sequences. RefSeq No. NP_003317.1 and Swiss-Prot Accession No. P23510-1 provide an exemplary human OX40L amino acid sequence for subtype 1. RefSeq No. NP_001284491.1 and Swiss-Prot Accession No. P23510-2 provide an exemplary human OX40L amino acid sequence for subtype 2. Human OX40L is designated by Entrez Gene as GeneID: 7292. In a specific embodiment, OX40L is human OX40L subtype 1 of SEQ ID NO:42 or subtype 2 of SEQ ID NO:43.

As used herein, the term "host cell" can be any type of cell (e.g., a primary cell, a cell in culture, or a cell from a cell line). In a specific embodiment, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of this cell may not be the same as the parent cell transfected with nucleic acid molecules The same because of, for example, mutations or environmental influences that may occur in the offspring or when the nucleic acid molecule is integrated into the host cell genome.

As used herein, in the context of administering treatment to a subject, the term "effective amount" refers to the amount of treatment that achieves the desired preventive or therapeutic effect. Examples of effective amounts are provided in section 5.5.1.3 below.

As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some embodiments, the subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human), most preferably Is human. In some embodiments, the subject is a cynomolgus monkey. In certain embodiments, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, cows, cats, or dogs). In some embodiments, such terms refer to pets or livestock. In specific embodiments, such terms refer to humans.

As used herein, if the binding between the test antibody and the first antigen is reduced by at least 30%, 40%, 50%, 60%, 70%, or 80% relative to the binding between the test antibody and the second antigen %, then the binding between the test antibody and the first antigen is "substantially weaker" relative to the binding between the test antibody and the second antigen, as measured, for example, in a flow cytometry analysis.

The determination of "percent identity" between two sequences (for example, amino acid sequence or nucleic acid sequence) can also be done using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S and Altschul SF (1990) Proceedings of the National Academy of Sciences 87: 2264-2268. The algorithm was modified in Karlin S and Alchure SF (1993) Proceedings of the National Academy of Sciences 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Alchure SF et al. (1990) Journal of Molecular Biology 215:403. The BLAST nucleotide search can be performed by setting the parameters of the NBLAST nucleotide program to, for example, score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein search can be performed by setting XBLAST program parameters to, for example, a score of 50 and word length=3 to obtain amino acid sequences that are homologous to the protein molecules described herein. In order to obtain gapped alignments for comparison targets, gapped BLAST as described in Alchure SF et al. (1997) Nucleic Acids Res. 25: 33893402 can be used. Alternatively, PSI BLAST can be used to perform iterative search ( Id. ) for detecting the distance relationship between molecules. When using BLAST, Gapped BLAST, and PSI Blast programs, you can use the default parameters of the corresponding programs (for example, XBLAST and NBLAST) (see, for example, National Biotechnology on the World Wide Web (ncbi.nlm.nih.gov) Information Center (National Center for Biotechnology Information, NCBI)). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller (1988, CABIOS 4:11 17). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, you can use the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

Techniques similar to those described above can be used to determine the percent identity between two sequences with or without gaps. In calculating percent agreement, only exact matches are typically counted.

As used herein, the term "an antigen-binding domain that does not bind to an antigen expressed by human immune cells" means that the antigen-binding domain does not bind to an antigen expressed by any human cell of hematopoietic origin that plays a role in an immune response. effect. Immune cells include lymphocytes (such as B cells and T cells); natural killer cells; and bone marrow cells (such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes). For example, such a binding domain will not bind to any other member of the OX40 or TNF receptor superfamily expressed by human cells. However, the antigen binding domain can bind to antigens such as but not limited to: antigens expressed in other organisms other than humans (ie non-human antigens); antigens not expressed by wild-type human cells; or viral antigens (including But not limited to antigens derived from viruses that do not infect human cells, or viral antigens that are not present in uninfected human immune cells).

5.2 Antibodies

In a specific aspect, provided herein are antibodies (e.g., monoclonal antibodies (e.g., chimeric, humanized, or human antibodies)) that specifically bind to OX40 (e.g., human OX40).

In certain embodiments, the antibodies described herein bind to human CD4+ T cells and human CD8+ T cells. In some embodiments In, the antibodies described herein bind to human CD4+ cells and cynomolgus CD4+ T cells.

In a specific embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include a light chain variable region (VL) that includes:

(a) VL CDR1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO:1), consisting of, or consisting essentially of,

(b) Including the amino acid sequence LGSNRAS (SEQ ID NO: 2), VL CDR2 consisting of, or consisting essentially of, and

(c) The VL CDR3 including the amino acid sequence MQALQTPLT (SEQ ID NO: 3), consisting of, or consisting essentially of the amino acid sequence MQALQTPLT (SEQ ID NO: 3), as shown in Table 1.

In some embodiments, the antibody includes the VL framework regions described herein. In a specific embodiment, the antibody includes the VL framework regions (FR) of the antibodies listed in Table 3.

In another embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include a heavy chain variable region (VH) that includes:

(a) VH CDR1 including the amino acid sequence GSAMH (SEQ ID NO: 4), consisting of, or consisting essentially of it,

(b) Including the amino acid sequence RIRSKANSYATAYAASVKG (SEQ ID NO: 5), VH CDR2 consisting of, or consisting essentially of, and

(c) Including the amino acid sequence GIYDSSGYDY (SEQ ID NO: 6) The VH CDR3 consisting of or essentially consisting of it is shown in Table 2.

In some embodiments, the antibody includes the VH framework described herein. In a specific embodiment, the antibody includes the VH framework regions of the antibodies listed in Table 4.

In a specific embodiment, the antibody includes the four VL framework regions (FR) listed in Table 3 and the four VH framework regions (FR) listed in Table 4.

In some embodiments, provided herein are antibodies that specifically bind to OX40 (eg, human OX40) and include the light chain variable region (VL) CDR and heavy chain variable region (VH) CDR of pab1949 or pab2044, such as , As listed in Tables 1 and 2 (ie SEQ ID NO: 1-6). In some embodiments, provided herein are antibodies that specifically bind to OX40 (eg, human OX40) and include the light chain variable region (VL) CDR and heavy chain variable region (VH) CDR of pab1949 or pab2044, such as , As listed in Tables 1 and 2 (ie SEQ ID NO: 1-6), and VL framework regions and VH framework regions are listed in Tables 3 and 4.

In a specific embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include a light chain variable region (VL) including: as listed in Table 1. VL CDR1, VL CDR2, and VL CDR3 and those listed in Table 3 VL framework area.

In certain embodiments, the antibody includes a light chain variable framework region derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence is the amino acid sequence of IGKV2-28*01 Sequence (SEQ ID NO: 18).

In a specific embodiment, the antibodies described herein that specifically bind to OX40 (for example, human OX40) include a heavy chain variable region (VH) including: as listed in Table 2 VH CDR1, VH CDR2, and VH CDR3 and the VH framework regions listed in Table 4.

In certain embodiments, the antibody includes a heavy chain variable framework region derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence is the amino acid sequence of IGHV3-73*01 Sequence (SEQ ID NO: 19).

In a specific embodiment, an antibody that specifically binds to OX40 (for example, human OX40) includes: a VL domain, which includes the amino acid sequence of SEQ ID NO:15. In a specific embodiment, an antibody that specifically binds to OX40 (e.g., human OX40) includes a VL domain consisting of, or essentially consisting of, the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an antibody that specifically binds to OX40 (eg, human OX40) includes: a VH domain, which includes the amino acid sequence of SEQ ID NO:16. In some embodiments, antibodies that specifically bind to OX40 (e.g., human OX40) include NO: 16 amino acid sequence consisting of, or a VH domain consisting essentially of it.

In certain embodiments, an antibody that specifically binds to OX40 (e.g., human OX40) includes a VH domain and a VL domain, wherein the VH domain and the VL domain include SEQ ID NO: 16 and SEQ ID NO: 15, respectively. Amino acid sequence. In certain embodiments, an antibody that specifically binds to OX40 (e.g., human OX40) includes a VH domain and a VL domain, wherein the VH domain and the VL domain are represented by SEQ ID NO: 16 and SEQ ID NO: 15, respectively. The amino acid sequence consists of, or consists essentially of, the sequence of amino acids.

In certain aspects, the antibodies described herein may be described in terms of their VL domain alone or in terms of their VH domain alone, or in terms of their 3 VL CDRs alone, or in terms of their 3 VH CDRs alone. See, for example, Rader C et al. (1998) Proceedings of the American Academy of Sciences 95:8910-8915, which is incorporated herein by reference in its entirety, which describes the humanization of mouse anti-αvβ3 antibodies , By identifying complementary light chains or heavy chains from the human light chain or heavy chain library, respectively, to obtain humanized antibody variants with affinity as high as or higher than that of the original antibody . See also Clarkson T et al. (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, which describes the use of specific VL domains (or VH domains) and A method of screening libraries for complementary variable domains to generate antibodies that bind to specific antigens. Screening produces 14 new partners for specific VH domains and 13 new partners for specific VL domains, these partners It is a strong binding agent, as determined by ELISA. See also Kim SJ and Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, which describes the use of specific VH domains and methods for screening libraries for complementary VL domains (e.g., human VL libraries) to generate antibodies that bind to specific antigens; the selected VL domains can then be used to guide the selection of additional complementary (e.g., human) VHs Domain.

In certain aspects, the CDR of an antibody can be determined according to the Chothia numbering scheme (which refers to the position of the immunoglobulin structural loop) (see, for example, Josia C and Lesk Am, (1987), Journal of Molecular Biology 196:901-917; Al-Lazikani B, et al., (1997) Journal of Molecular Biology 273:927-948; Josia C, et al., (1992) Journal of Molecular Biology 227:799 -817; Tramontano A et al. (1990) Journal of Molecular Biology 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when the Kabat numbering convention is used, the Chothia CDR-H1 loop is present in the heavy chain amino acid 26 to 32, 33 or 34, and the Chothia CDR-H2 loop is present in the heavy chain amino acid 52 To 56, and the Chothia CDR-H3 loop is present in the heavy chain amino acid 95 to 102, while the Chothia CDR-L1 loop is present in the light chain amino acid 24 to 34, and the Chothia CDR-L2 loop is present in the light chain amine The base acid is 50 to 56 and the Chothia CDR-L3 loop is present at 89 to 97 of the light chain amino acid. When using the Kabat numbering rules for numbering, the end of the Chothia CDR-H1 loop varies between H32 and H34 according to the length of the loop (this system Because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B exists, the ring terminates at 32; or if only 35A exists, the ring terminates at 33; if both 35A and 35B exist, Then the ring terminates at 34).

In certain aspects, provided herein are antibodies that specifically bind to the Chothia VL CDRs of OX40 (eg, human OX40) and include the VL of pab1949 or pab2044. In certain aspects, provided herein are antibodies that specifically bind to the Chothia VH CDR of the VH of OX40 (eg, human OX40) and include pab1949 or pab2044. In certain aspects, provided herein are antibodies that specifically bind to OX40 (eg, human OX40) and include the Chothia VL CDR of the VL of pab1949 or pab2044 and the Chothia VH CDR of the VH of pab1949 or pab2044. In certain embodiments, an antibody that specifically binds to OX40 (e.g., human OX40) includes one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, provided herein is an antibody that specifically binds to OX40 (e.g., human OX40) and includes a combination of Kabat CDR and Chothia CDR.

In certain aspects, the CDR of an antibody can be based on as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP et al., (1999) Nucleic Acid Research (Nucleic Acids Res) 27: 209-212 IMGT numbering system to determine. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, and VL- CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In a specific embodiment, provided herein are antibodies that specifically bind to OX40 (e.g., human OX40) and include the CDRs of pab1949 or pab2044, such as by the IMGT numbering system (e.g., as described in the above-mentioned Lefranc MP (1999 ) And the above-mentioned LeFran MP et al. (1999)).

In certain aspects, the CDR of an antibody can be determined according to MacCallum RM et al. (1996) Journal of Molecular Biology 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains" in "Antibody Engineering", Kangte Kontermann and Dübel, editors, Chapter 31, pages 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, an antibody that specifically binds to OX40 (for example, human OX40) and includes the CDR of pab1949 or pab2044 is provided herein, as determined by the method in McAllen RM et al.

In some aspects, the CDR of an antibody can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, which represents the balance between the Kabat CDR and the Chothia structural loop, and is recognized by Oxford Used by Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, here is provided An antibody that specifically binds to OX40 (e.g., human OX40) and includes the CDR of pab1949 or pab2044, as determined by the AbM numbering scheme.

In a specific embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), the position of one or more CDRs along the VH (e.g., CDR1, CDR2 or CDR3) and/or VL (e.g., CDR1, CDR2 or CDR3) regions of the antibodies described herein can vary by one or two , Three, four, five or six amino acid positions. For example, in one embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%). %, at least 95%), relative to the CDR position of the antibody described herein (for example, pab1949 or pab2044), the position of the CDR of the antibody described herein can be defined by shifting the N-terminal and/or C-terminal boundary of the CDR The position is one, two, three, four, five or six amino acids. In another embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regions of the antibodies described herein can vary (e.g., (Shorter or longer) one, two, three, four, five or more amino acids.

In one embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, At least 95%), the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may be more than one or more CDRs described herein (e.g., SEQ ID NO: 1-6 ) Short one, two, three, four, five or more amino acids. In another embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may be more than one or more CDRs described herein (e.g., SEQ ID NO: 1- 6) Long one, two, three, four, five or more amino acids. In another embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), the amino ends of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be compared to one or more of the CDRs described herein (e.g., SEQ ID NO: 1-6) extend one, two, three, four, five or more amino acids. In another embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), VL CDR1, VL described here The carboxyl-terminus of CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 may be extended by one, two, three, compared to one or more of the CDRs described herein (e.g., SEQ ID NO: 1-6) Four, five or more amino acids. In another embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , At least 95%), the amino ends of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be compared to one or more of the CDRs described herein (e.g., SEQ ID NO: 1-6) Shorten one, two, three, four, five or more amino acids. In one embodiment, as long as the immunospecific binding to OX40 (e.g., human OX40) can be maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, At least 95%), the carboxy terminus of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be compared to one or more CDRs described herein (e.g., SEQ ID NO: 1-6) Shorten one, two, three, four, five or more amino acids. Any method known in the art can be used to confirm whether immunospecific binding to OX40 (e.g., human OX40) is maintained, for example, the binding analysis described in the "Example" section (Chapter 6) and condition.

In specific aspects, provided herein are antibodies that include antibody light and heavy chains (e.g., light and heavy chains, respectively). Regarding the light chain, in a specific embodiment, the antibody light chain described herein is a kappa light chain. In another specific embodiment, the antibody light chain described herein is a lambda light chain. exist In yet another specific embodiment, the antibody light chain described herein is a human kappa light chain or a human lambda light chain. In a specific embodiment, the antibodies described herein that immunospecifically bind to an OX40 polypeptide (e.g., human OX40) include a light chain, wherein the amino acid sequence of the VL domain includes the sequence listed in SEQ ID NO: 15 And wherein the constant region of the light chain includes the amino acid sequence of the constant region of the human kappa light chain. In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (eg, human OX40) include a light chain, wherein the amino acid sequence of the VL domain includes the sequence listed in SEQ ID NO: 15 And the constant region of the light chain includes the amino acid sequence of the constant region of the human lambda light chain. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a light chain, wherein the amino acid sequence of the VL domain includes the sequence listed in SEQ ID NO: 15, And the constant region of the light chain includes the amino acid sequence of the constant region of the human kappa or lambda light chain. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and the aforementioned Kabat EA et al. (1991).

In a specific embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a light chain that includes the amino acid sequence set forth in SEQ ID NO:20.

Regarding the heavy chain, in a specific embodiment, the antibody heavy chain described herein may be an alpha, delta, epsilon, gamma, or mu heavy chain. In another specific embodiment, the antibody heavy chain may include a human alpha, delta, epsilon, gamma, or mu heavy chain. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a heavy chain, wherein The amino acid sequence of the VH domain may include the sequence listed in SEQ ID NO: 16, and the constant region of the heavy chain includes the amino acid sequence of the human gamma heavy chain constant region. In a specific embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include a heavy chain, wherein the amino acid sequence of the VH domain includes the sequence listed in SEQ ID NO: 16, and The constant region of the heavy chain includes the amino acids of the human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and the aforementioned Kabat EA et al. (1991).

In a specific embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:21. In a specific embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:60. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:23. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:61. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:51. In another embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include a heavy chain, which is included in SEQ ID NO: The amino acid sequence listed in 62. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:52. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include a heavy chain that includes the amino acid sequence set forth in SEQ ID NO:63.

In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include VL domains and VH domains comprising any of the amino acid sequences described herein, and wherein the constant region includes IgG , IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, or the amino acid sequence of the constant region of human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules. In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include VL domains and VH domains comprising any of the amino acid sequences described herein, and wherein the constant region includes IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, any class (e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ ) or any subclass (e.g., IgG _2a And IgG _2b ) the amino acid sequence of the constant region of the immunoglobulin molecule. In a specific embodiment, the constant region includes human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, of any kind (e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ ) The amino acid sequence of the constant region of an immunoglobulin molecule or any subclass (for example, IgG _2a and IgG _2b).

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include VL domains and VH domains comprising any of the amino acid sequences described herein, and wherein the constant region includes human IgG ₁ (e.g., allotype G1m3, G1m17,1 or G1m17,1,2), human IgG _2, or the amino acid sequence of human IgG ₄ constant region. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include VL domains and VH domains comprising any of the amino acid sequences described herein, and wherein the constant region includes human The amino acid sequence of the constant region of IgG _{1 (allogeneic G1m3).} Non-limiting examples of human constant regions are described in the art, for example, see Kabat EA et al., (1991) above.

In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: a light chain including the amino acid sequence listed in SEQ ID NO: 20, and a heavy chain The heavy chain includes the amino acid sequence listed in SEQ ID NO:21. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: a light chain including the amino acid sequence listed in SEQ ID NO: 20, and a heavy chain The heavy chain includes the amino acid sequence listed in SEQ ID NO:60. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: a light chain including the amino acid sequence listed in SEQ ID NO: 20, and a heavy chain The heavy chain includes the amino acid sequence listed in SEQ ID NO:23. In another embodiment, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include: a light chain that includes the amino acid sequence listed in SEQ ID NO: 20, and The heavy chain includes the amino acid sequence listed in SEQ ID NO:61. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: a light chain including the amino acid sequence listed in SEQ ID NO: 20, and a heavy chain The heavy chain includes the amino acid sequence listed in SEQ ID NO: 51 or 52. In another embodiment, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: a light chain including the amino acid sequence listed in SEQ ID NO: 20, and a heavy chain The heavy chain includes the amino acid sequence listed in SEQ ID NO: 62 or 63.

In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibodies described herein (e.g., CH2 domain (residues 231 to 340 of _{human IgG 1)} And/or CH3 domain ( _{residues 341-447 of human IgG 1} ) and/or hinge region, which have a numbering according to the Kabat numbering system (for example, the EU index in Kabat), To change one or more functional properties of the antibody (such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cytotoxicity).

In some embodiments, one, two or more mutations (e.g., amino acid substitution) are introduced into the hinge region of the Fc region (CH1 domain), so that the cysteine residues in the hinge region The number of bases is changed (e.g., increased or decreased), as described in, for example, U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be changed, for example, to assist in the assembly of light and heavy chains, or to change (e.g., increase or decrease) the stability of the antibody.

In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231 to 340 of _{human IgG 1) and} / Or CH3 domain ( _{residues 341-447 of human IgG 1} ) and/or hinge region, which have a numbering according to the Kabat numbering system (for example, the EU index in Kabat) to Increase or decrease the affinity of the antibody for Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Mutations in the Fc region of antibodies that decrease or increase the affinity of the antibody for Fc receptors and for the use of such The technology for introducing mutations into Fc receptors or fragments thereof is known to those skilled in the art. Examples of mutations in the Fc receptor of antibodies that can change the affinity of the antibody for Fc receptors are described in, for example, Smith (Smith ) P et al. (2012) Proceedings of the National Academy of Sciences 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication No. WO 02/060919; WO 98/23289; and WO 97/34631. The reference is incorporated here.

In a specific embodiment, one, two or more amino acid mutations (ie, substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably, Fc or hinge-Fc structure Domain fragments) to change (e.g., reduce or increase) the half-life of antibodies in vivo. For examples of mutations that will alter (e.g., decrease or increase) the half-life of antibodies in vivo, see, e.g., International Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Patent Nos. 5,869,046, 6,121,022 , 6,277,375 and 6,165,745. In some embodiments, one, two or more amino acid mutations (ie, substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably, Fc or hinge-Fc domain Fragments) to reduce the half-life of antibodies in the body. In other embodiments, one, two or more amino acid mutations (ie, substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably, Fc or hinge-Fc domain Fragments) to increase the half-life of antibodies in vivo. In a specific embodiment, the antibody can be in the second constant (CH2) domain ( _{residues 231 to 340 of human IgG 1} ) and/or the third constant (CH3) domain (residues 341-447 of _{human IgG 1).} ) Has one or more amino acid mutations (for example, substitutions), which have a number according to the EU index in Kabat (Kabat EA et al., (1991) above). In a specific embodiment, _{the constant region of the IgG 1} of the antibody described herein includes the substitution of methionine (M) at position 252 with tyrosine (Y) and the substitution of serine (S) at position 254 with threon. Amino acid (T) and threonine (T) at position 256 are substituted with glutamine (E), numbered according to the EU index in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (called "YTE mutant") has been shown to exhibit a four-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) Journal of Biological Chemistry 281: 23514-24). In certain embodiments, the antibody includes an IgG constant domain that includes one, two, and three amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436. Or more amino acid substitutions are numbered according to the EU index in Kabat.

In other embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of an IgG constant domain to change one or more effector functions of the antibody. For example, one or more amines selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 (numbered according to the EU index in Kabat) The base acid is replaced with a different amino acid, so that the antibody has an altered affinity for the effector ligand, but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domains (via point mutations or other means) can reduce the Fc receptor binding of circulating antibodies, thereby improving tumor localization. For a description of mutations that delete or inactivate constant domains and thereby increase tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In certain embodiments, one or more amino acid substitutions can be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region that can reduce Fc receptor binding (see, For example, Shields RL et al. (2001) Journal of Biological Chemistry 276: 6591-604). In different embodiments, one or more of the following mutations can occur in the constant region of the antibodies described herein: N297A substitution; N297Q substitution; L235A substitution and L237A substitution; L234A substitution and L235A substitution; E233P substitution; L234V substitution ; L235A substitution; C236 deletion; P238A substitution; D265A substitution; A327Q substitution; or Replaced by P329A and numbered according to the EU index in Kabat. In certain embodiments, mutations selected from the group consisting of D265A, P329A, and combinations thereof may occur in the constant region of the antibodies described herein.

In a specific embodiment, the antibodies described herein include the constant domain _{of IgG 1 with N297Q or N297A amino acid substitutions.} In one embodiment, the antibodies described herein include _{the constant domain of IgG 1} with mutations selected from the group consisting of D265A, P329A, and combinations thereof.

In some embodiments, one or more of the constant regions of the antibodies described herein can be selected from amino acid residues 329, 331, and 322 (according to the EU index in Kabat). (EU index) for numbering) Substituting different amino acids so that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in US Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues within the amino acid positions 231 to 238 in the N-terminal region of the CH2 domain of the antibody described herein can be changed to thereby change the antibody The ability to fix complement. This approach is further described in International Publication No. WO 94/29351. In some embodiments, by mutating one or more amino acids at the following positions (for example, introducing amino acid substitutions), the Fc region of the antibody described herein is modified to increase the antibody's resistance to Body-dependent cellular cytotoxicity (ADCC) to mediate, and/or to increase the antibody’s ability to target a Fcγ receptor affinity: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289 , 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334 , 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, according to Kabat In the EU index (EU index) for numbering. This approach is further described in International Publication No. WO 00/42072.

In certain embodiments, the antibodies described herein include mutations (e.g., substitutions) at positions 267, 328, or a combination thereof (numbering according to the EU index in Kabat) The constant domain of IgG _{1. In certain embodiments, the antibodies described herein include the constant domain of IgG 1} with a mutation (e.g., substitution) selected from the group consisting of S267E, L328F, and combinations thereof. In certain embodiments, the antibodies described herein include the constant domain _{of IgG 1} with S267E/L328F mutations (e.g., substitutions). _{In certain embodiments, antibodies described herein that include the constant domain of IgG 1} with S267E/L328F mutations (eg, substitutions) have increased binding affinity for FcyRIIA, FcyRIIB, or FcyRIIA and FcyRIIB.

In certain embodiments, the antibodies described herein include _{the constant domain of an IgG 4} antibody, and a serine at residue 228 of the heavy chain amino acid (according to the EU index in Kabat) Numbering) is substituted with proline.

In certain embodiments, the antibodies described herein include _{the constant domain of an IgG 2} antibody, and the cysteine at residue 127 of the heavy chain amino acid residue (numbering according to Kabat) is substituted with serine acid.

It has been reported that antibodies with low fucose content have increased affinity for Fc receptors (e.g., FcyRIIIA). Therefore, in certain embodiments, the antibodies described herein have low fucose content or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, antibodies can be expressed in cells that are defective in fucosylation or lack the ability to fucosylate. In a specific example, a cell line with two alleles knocking out α1,6-fucosyltransferase can be used to produce antibodies with low fucose content. The Potelligent ^® system (Lonza) is an example of such a system and can be used to produce antibodies with low fucose content. Alternatively, antibodies with low or no fucose content can be produced, for example, by: (i) culturing the cells under conditions that avoid or reduce fucosylation; (ii) remove after translation Fucose (for example, with fucosidase); (iii) adding the desired sugar after translation (for example, after recombining a glycoprotein that exhibits non-glycosylation); or (iv) purifying the glycoprotein to select non-glycosylated glycoproteins Fucosylated antibody. For methods for producing antibodies with no fucose content or low fucose content, see, for example, Longmore GD and Schachter H (1982) Carbohydr Res 100 : 365-92 and Imai-Nishiya H et al. (2007) BMC Biotechnol. 7:84.

The engineered glycoforms can be used for a variety of purposes, including, but not limited to, enhancing or reducing effector functions. Methods for generating engineered glycoforms in the antibodies described herein include, but are not limited to, disclosed in, for example, Umaña P et al. (1999) Nat Biotechnol 17:176-180; Davies J et al. (2001) Biotechnol Bioeng 74: 288-294; Shields RL et al. (2002) Journal of Biological Chemistry 277: 26733-26740; Shinkawa T et al. (2003) Journal of Biological Chemistry 278: 3466-3473; Niwa R et al. (2004) Clin Cancer Res 1: 6248-6255; Presta ( Presta) LG et al. (2002) Biochem Soc Trans 30: 487-490; Kanda Y et al. (2007) Glycobiology 17: 104-118; USA Patent Case No. 6,602,684; 6,946,292; and 7,214,775; US Patent Publication No. US 2007/0248600; 2007/0178551; 2008/0060092; and 2006/0253928; International Publication No. WO 00/61739; WO 01/292246; WO 02/ 311140; and WO 02/30954; Potillegent ^™ technology (Biowa, Princeton, New Jersey (NJ)); and GlycoMAb® glycosylation engineering technology (Glycart Biotechnology AG, Zurich, Switzerland). See also, for example, Ferrara C et al. (2006) Biotechnology and Bioengineering 93:851-861; International Publication No. WO 07/039818; WO 12/130831; WO 99/054342; WO 03 /011878; and WO 04/065540.

In certain embodiments, the engineered antibody used as described herein Technology Xencor (Monrovia, CA) an Fc domain Xmab ^® technology. See, for example, U.S. Patent No. 8,367,805; 8,039,592; 8,124,731; 8,188,231; U.S. Patent Publication No. 2006/0235208; International Publication No. WO 05/077981; WO 11/097527; and Richards JO et al., (2008) Mol Cancer Ther 7: 2517-2527.

In certain embodiments, the amino acid residues at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain in the constant region of the antibody described herein (according to the numbered EU index (EU index) Numbering) is not L, L, and D respectively. This approach is described in detail in International Publication No. WO 14/108483. In a specific embodiment, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively.

In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both of the heavy chain constant regions of an antibody having two heavy chain constant regions described herein.

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, which is included in SEQ ID NO: VL CDR1, VL CDR2, and VL CDR3 amino acid sequences listed in 1-3 (for example, those listed in Table 1); (ii) The heavy chain includes a VH domain, which includes The VH CDR1, VH CDR2, and VH CDR3 amino acid sequences listed in SEQ ID NO: 4-6 (for example, those listed in Table 2); (iii) the light chain further includes a light chain constant domain, The light chain constant domain includes the amino acid sequence of the human kappa light chain constant domain; and (iv) the heavy chain further includes a heavy chain constant domain, and the heavy chain constant domain includes human IgG ₁ (optionally IgG ₁ (allogeneic G1m3) )) The amino acid sequence of the constant domain of the heavy chain.

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, which is included in SEQ ID NO; the amino acid listed in 15; (ii) the heavy chain includes the VH domain, which domain includes the amino acid sequence listed in SEQ ID NO: 16; (iii) the light chain further includes A constant domain including the amino acid sequence of the constant domain of a human kappa light chain; and (iv) the heavy chain further includes a constant domain including a human IgG ₁ (optionally IgG ₁ (allogeneic G1m3)) heavy The amino acid sequence of the constant domain of the chain.

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, which is included in SEQ ID NO: VL CDR1, VL CDR2, and VL CDR3 amino acid sequences listed in 1-3 (for example, those listed in Table 1); (ii) the heavy chain includes a VH domain, which includes The VH CDR1, VH CDR2, and VH CDR3 amino acid sequences listed in SEQ ID NO: 4-6 (for example, those listed in Table 2); (iii) the light chain further includes a light chain constant domain, The light chain constant domain includes the amino acid sequence of a human kappa light chain constant domain; and (iv) the heavy chain further includes a heavy chain constant domain, and the heavy chain constant domain includes the amino acid sequence of a _{human IgG 4 heavy chain constant domain} .

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (eg, human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, and the domain includes SEQ ID NO: 15 amino acid sequence; (ii) the heavy chain includes a VH domain, which domain includes the amino acid sequence of SEQ ID NO: 16; (iii) the light chain further includes a constant domain, the constant domain includes The amino acid sequence of the constant domain of the human kappa light chain; and (iv) the heavy chain further includes a constant domain including the amino acid sequence of _{the constant domain of the human IgG 4 heavy chain.}

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, which is included in SEQ ID NO: VL CDR1, VL CDR2, and VL CDR3 amino acid sequences listed in 1-3 (for example, those listed in Table 1); (ii) the heavy chain includes a VH domain, which includes The VH CDR1, VH CDR2, and VH CDR3 amino acid sequences listed in SEQ ID NO: 4-6 (for example, those listed in Table 2); (iii) the light chain further includes a light chain constant domain, The light chain constant domain includes the amino acid sequence of a human kappa light chain constant domain; and (iv) the heavy chain further includes a heavy chain constant domain, and the heavy chain constant domain includes the amino acid sequence of a _{human IgG 2 heavy chain constant domain} .

In another specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (eg, human OX40) include a light chain and a heavy chain, wherein (i) the light chain includes a VL domain, and the domain includes SEQ ID NO: 15 amino acid sequence; (ii) the heavy chain includes a VH domain, which domain includes the amino acid sequence of SEQ ID NO: 16; (iii) the light chain further includes a constant domain, the constant domain includes The amino acid sequence of the constant domain of the human kappa light chain; and (iv) the heavy chain further includes a constant domain including the amino acid sequence of _{the constant domain of the human IgG 2 heavy chain.} In certain embodiments, the light chain includes the amino acid sequence of SEQ ID NO:50 and the heavy chain includes the amino acid sequence of SEQ ID NO:51. In certain embodiments, the light chain includes the amino acid sequence of SEQ ID NO:50 and the heavy chain includes the amino acid sequence of SEQ ID NO:62. In certain embodiments, the light chain includes the amino acid sequence of SEQ ID NO: 20 and the heavy chain includes the amino acid sequence of SEQ ID NO: 51. In certain embodiments, the light chain includes the amino acid sequence of SEQ ID NO: 20 and the heavy chain includes the amino acid sequence of SEQ ID NO: 62.

In another specific embodiment, the antibody that specifically binds to OX40 (for example, human OX40) provided herein includes: (a) a heavy chain including the amino acid sequence of SEQ ID NO: 21, which is at the amino acid position An amino acid substitution with N substitution to A or Q at 297; and (b) a light chain including the amino acid sequence of SEQ ID NO:20. In another specific embodiment, the antibody that specifically binds to OX40 (for example, human OX40) provided herein includes: (a) a heavy chain including the amino acid sequence of SEQ ID NO: 60, which is at the amino acid position An amino acid substitution with N substitution to A or Q at 297; and (b) a light chain including the amino acid sequence of SEQ ID NO:20.

In another specific embodiment, the antibody that specifically binds to OX40 (for example, human OX40) provided herein includes: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, which has a heavy chain selected from the group consisting of The group consists of the following: substitution of S to E at position 267 of the amino acid, substitution of L to F at position 328 of the amino acid, and substitution of S to E at position 267 of the amino acid And the substitution of L to F at position 328 of the amino acid; and (b) a light chain including the amino acid sequence of SEQ ID NO:20. In another specific embodiment, the antibody that specifically binds to OX40 (for example, human OX40) provided herein includes: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 60, which has a heavy chain selected from the group consisting of The group consists of the following: substitution of S to E at position 267 of the amino acid, substitution of L to F at position 328 of the amino acid, and substitution of S to E at position 267 of the amino acid And the substitution of L to F at position 328 of the amino acid; and (b) a light chain including the amino acid sequence of SEQ ID NO:20.

In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity. In specific embodiments, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) trigger natural killer cell-mediated cell depletion. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) are used to treat tumors infiltrated by natural killer cells. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) exhibit antibody-dependent cellular phagocytosis (ADCP) active. In specific embodiments, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) trigger macrophage-mediated cell depletion. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) are used to treat tumors infiltrated by macrophages. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) selectively deplete regulatory T cells in the tumor.

In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (for example, human OX40) include a framework region (for example, a framework region of a VL domain and/or a VH domain), which is a human framework region Or derived from the human framework area. Non-limiting examples of human framework regions are described in the art, for example, see Kabat EA et al ., (1991) above. In certain embodiments, the antibodies described herein include a framework region (eg, a framework region of a VL domain and/or a VH domain) that is a primate (eg, non-human primate) framework region Or derived from the primate (e.g., non-human primate) framework region.

For example, CDRs from antigen-specific non-human antibodies (typically of rodent origin (e.g., mouse or rat)) are grafted into a homologous human or non-human primate recipient framework. In one embodiment, the non-human primate receptor framework is from Old World ape. In a specific embodiment, the Old World ape (Old World ape) receptor framework is from a chimpanzee ( Pan troglodytes ), a bonobo ( Pan paniscus ) or a gorilla ( Gorilla gorilla ). In a specific embodiment, the non-human primate receptor framework is from a chimpanzee ( Pan troglodytes ). In a specific embodiment, the non-human primate receptor framework is an old world monkey receptor framework. In a specific embodiment, the old world monkey acceptor framework is from the genus Rhesus. In one embodiment, the non-human primate receptor framework is derived from cynomolgus monkey ( Macaca cynomolgus ). The non-human primate framework sequence is described in US Patent Application Publication No. US 2005/0208625.

In certain embodiments, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include one, two, or more VL framework regions (FR) that are directed against the antibodies listed in Table 3 above. The amino acid sequence described here. In some embodiments, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include one, two or more VH framework regions (FR) that have specificity against the antibodies listed in Table 4 above. The amino acid sequence described here. In a specific embodiment, the antibodies described herein that specifically bind to OX40 (for example, human OX40) include: one, two, or more VL framework regions, the framework regions having antibodies against the antibodies listed in Table 3 above are described here The amino acid sequence of, and one, two or more VH framework regions having the amino acid sequences described herein for the antibodies listed in Table 4 above.

In some embodiments, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include: one, two, three, or four framework regions of a VL domain having pab1949 or pab2044 (e.g. , SEQ ID NO: 7-10) amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid mutations (for example, amino acid substitution (Such as conservative amino acid substitution)) and/or VH structure The framework region of a domain that has the amino acid sequence of pab1949 or pab2044 (for example, SEQ ID NO: 11-14). In certain embodiments, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include: one, two, three, or four framework regions of a VH domain having pab1949 or pab2044 ( For example, the amino acid sequence of SEQ ID NO: 11-14), which has 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid mutations (e.g., amino acid Substitution (such as conservative amino acid substitution)) and/or the framework region of the VL domain, which has the amino acid sequence of pab1949 or pab2044 (for example, SEQ ID NO: 7-10).

In certain embodiments, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include at least 70%, at least 75%, at least 80%, or at least the VL framework region described herein in Table 3 above. A VL framework region (FR) with 85%, at least 90%, at least 95%, or at least 98% sequence identity. In certain embodiments, the antibodies described herein that specifically bind to OX40 (e.g., human OX40) include at least 70%, at least 75%, at least 80%, or at least the VH framework regions described herein in Table 4 above. 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH framework region (FR). In some embodiments, the antibodies described herein that specifically bind to OX40 (eg, human OX40) include: at least 70%, at least 75%, at least 80%, at least the VH framework region described herein in Table 4 above 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH framework region (FR), and the VL framework region described here in Table 3 above has at least 70%, at least 75%, A VL framework region (FR) with at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity.

The determination of the percent identity between two sequences (for example, amino acid sequence or nucleic acid sequence) can also be done using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S and Altschul SF (1990) Proceedings of the National Academy of Sciences 87: 2264-2268. The algorithm was modified in Karlin S and Alchure SF (1993) Proceedings of the National Academy of Sciences 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Alchure SF et al. (1990) Journal of Molecular Biology 215:403. The BLAST nucleotide search can be performed by setting the parameters of the NBLAST nucleotide program to, for example, score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein search can be performed by setting XBLAST program parameters to, for example, a score of 50 and word length=3 to obtain amino acid sequences that are homologous to the protein molecules described herein. In order to obtain gapped alignments for comparison targets, gapped BLAST as described in Alchure SF et al. (1997) Nucleic Acids Res. 25: 3389 3402 can be used. Alternatively, PSI BLAST can be used to perform iterative search ( Id. ) for detecting the distance relationship between molecules. When using BLAST, Gapped BLAST, and PSI Blast programs, you can use the default parameters of the corresponding programs (for example, XBLAST and NBLAST) (see, for example, National Biotechnology on the World Wide Web (ncbi.nlm.nih.gov) Information Center (National Center for Biotechnology Information, NCBI)). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller (1988, CABIOS 4:11 17). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, you can use the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

Techniques similar to those described above can be used to determine the percent identity between two sequences with or without gaps. In calculating percent agreement, only exact matches are typically counted.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of a VL domain, wherein the antibody comprises a VL CDR identical to that of pab1949 or pab2044.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of a VH domain, wherein the antibody includes a VH CDR that is the same as the VH CDR of pab1949 or pab2044.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody includes the same VL CDR and VH CDR as those of pab1949 or pab2044.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein the antibody is compatible with staphylococcal enterotoxin A (SEA) (e.g., 100ng/ml) in combination, when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for 5 days, for example, the production of IL-2 in PBMC is induced, as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company)), where the IL-2 production line is, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ A substantial increase in antibody concentration between ml and 20 μg/ml, 4 μg/ml and 20 μg/ml, 0.032 μg/ml and 4 μg/ml, 0.16 μg/ml and 4 μg/ml, or 0.8 μg/ml and 4 μg/ml The function of sex. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein the antibody is combined with staphylococcal enterotoxin A (SEA) , Induce the production of IL-2 in, for example, PBMC, where the production of IL-2 is, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml And 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml in a substantially increasing function of the antibody concentration, as in, for example, the following Evaluation in the step of the assay: (a) In the absence or presence of different concentrations of the antibody (for example, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100 ng/ml SEA , such culture PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example, at e.g. 37 ℃, 5% CO _2, and 97% humidity; and (b) the supernatant was collected and clarified by the electrochemical e.g. Bioluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) measures IL-2 titer.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of a VL domain, wherein the antibody is compatible with Staphylococcal Enterotoxin A (SEA) (e.g., 100ng/ml) in combination, when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for 5 days, for example, the production of IL-2 in PBMC is induced, as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company)), where the anti-OX40 antibody is measured at 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and When between 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml, the production of IL-2 shows S Shaped dose response curve. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein the antibody is combined with staphylococcal enterotoxin A (SEA) , Induce the production of IL-2 in PBMC, for example, when the anti-OX40 antibody is at 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg /ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as in, for example, including Assess in the assay of the following steps: (a) In the absence or presence of different concentrations of the antibody (for example, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100ng/ml SEA lower, e.g. cultured at 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. Electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) measures IL-2 titer.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody is compatible with Staphylococcal Enterotoxin A (SEA) (e.g., 100ng/ml) in combination, when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for 5 days, for example, the production of IL-2 in PBMC is induced, as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company)), where IL-2 production is measured at, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ A substantial increase in antibody concentration between ml and 20 μg/ml, 4 μg/ml and 20 μg/ml, 0.032 μg/ml and 4 μg/ml, 0.16 μg/ml and 4 μg/ml, or 0.8 μg/ml and 4 μg/ml The function of sex. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody is combined with staphylococcal enterotoxin A (SEA) , Induce the production of IL-2 in, for example, PBMC, where the production of IL-2 is, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml And 20 μg/ml, 0.032 μg/ml and 4 μg/ml, 0.16 μg/ml and 4 μg/ml, or 0.8 μg/ml and 4 μg/ml in a substantially increasing function of the antibody concentration, as in, for example, the following Evaluation of the steps: (a) In the absence or presence of different concentrations of the antibody (for example, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100 ng/ml SEA , such culture PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example, at e.g. 37 ℃, 5% CO _2, and 97% humidity; and (b) the supernatant was collected and clarified by the electrochemical e.g. Bioluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) measures IL-2 titer.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody is compatible with Staphylococcal Enterotoxin A (SEA) (e.g., 100ng/ml) in combination, when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for 5 days, for example, the production of IL-2 in PBMC is induced, as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company)), where the anti-OX40 antibody is measured at 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and When between 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml, the production of IL-2 shows S Shaped dose response curve. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody is combined with staphylococcal enterotoxin A (SEA) , Induce the production of IL-2 in PBMC, for example, when the anti-OX40 antibody is at 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg /ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml, the production of IL-2 shows a sigmoidal dose response curve, as in, for example, including Evaluation in the following steps: (a) In the absence or presence of different concentrations of the antibody (for example, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100ng/ml SEA lower, e.g. cultured at 37 ℃, 5% CO _2, 97% humidity, and these PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. Electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) measures IL-2 titer.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (for example, 100ng/ml), when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for, for example, 5 days, induces, for example, PBMC The production of IL-2 is measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where the production of IL-2 is, for example, 0.032 μg/ml And 20μg/ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or A substantially increasing function of antibody concentration between 0.8 μg/ml and 4 μg/ml. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, in combination with staphylococcal enterotoxin A (SEA), induces, for example, the production of IL-2 in PBMC, where the production of IL-2 is, for example, 0.032μg/ml and 20μg/ml, 0.16μg/ml And 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml A substantially increasing function of antibody concentration between, as evaluated in an assay that includes, for example, the following steps: (a) In the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064 , 0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml of such culture PBMC (e.g., in the wells at 10 ⁵ cells e.g. 37 ℃, 5% CO _2, and 97% humidity) for e.g. 5 days; and (b) Collect the clear supernatant and measure the IL-2 titer by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, in combination with staphylococcal enterotoxin A (SEA) (for example, 100ng/ml), when _{stimulated at 37°C, 5% CO 2} , and 97% humidity for, for example, 5 days, induces, for example, PBMC The production of IL-2, as measured by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)), where anti-OX40 antibody is at 0.032 μg/ml and 20 μg /ml, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg When it is between 4μg/ml and 4μg/ml, the production of IL-2 shows a sigmoidal dose response curve. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, in combination with staphylococcal enterotoxin A (SEA), induces the production of IL-2 in PBMC, for example, when the anti-OX40 antibody is at 0.032μg/ml and 20μg/ml, 0.16μg/ml and 20μg /ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml, or 0.8μg/ml and 4μg/ml The production of IL-2 shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml, and these PBMC cultured at e.g. 37 ℃, 5% CO _2, and 97% humidity (e.g., in the well ¹⁰⁵ cells) Length For example, 5 days; and (b) Collect the clear supernatant and measure the IL-2 titer by, for example, electrochemiluminescence (for example, human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)).

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein when the antibody binds to the plate, the anti-CD3 antibody that binds to the plate (E.g., 0.8μg/ml) in combination, when _{stimulated at 37°C and 5% CO 2} for e.g. 4 days, one or more cytokines (e.g., TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human Primate (NHP) V-Plex assay kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, Or IL-13), for example, between 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml and 50 μg/ml, or 6.3 μg/ml and 50 μg/ml. A substantially increasing function of concentration, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (e.g., 0.8 μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml) in the presence of the antibody of the present invention bound to the plate, for example at 37°C and 5 Culturing these PBMCs under% CO _{2 for} , for example, 4 days; and (b) collecting the supernatant and performing electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso-Discovery Company)) or non- Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL) -13) Generation. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein when the antibody binds to the plate, the anti-CD3 antibody that binds to the plate (E.g., 0.8μg/ml) in combination, when _{stimulated at 37°C and 5% CO 2} for e.g. 4 days, one or more cytokines (e.g., TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), where the concentration of anti-OX40 antibody is, for example, 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg /ml and 50μg/ml, or between 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL- The production of 13) shows a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody (e.g., 0.8 μg/ml) bound on the plate and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50 μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) in the presence of the antibody of the present invention bound to the plate, for example, 37°C And culturing these PBMCs under 5% CO _{2 for} , for example, 4 days; and (b) Collect the supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) Or non-human primate (NHP) V-Plex assay kit (Meso Discovery Company)) to measure one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, Or IL-13).

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein when the antibody binds to the plate, the anti-CD3 antibody that binds to the plate (E.g., 0.8 μg/ml) in combination, when the _{stimulation is continued at, for example, 37°C and 5% CO 2 for} , for example, 4 days, one or more cytokines (e.g., TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, Or IL-13), for example, between 0.7 μg/ml and 50 μg/ml, 1.6 μg/ml and 50 μg/ml, 3.1 μg/ml and 50 μg/ml, or 6.3 μg/ml and 50 μg/ml. A substantially increasing function of concentration, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (e.g., 0.8 μg/ml) and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml) in the presence of the antibody of the present invention bound to the plate, for example at 37°C and 5 Culturing these PBMCs under% CO _{2 for} , for example, 4 days; and (b) collecting the supernatant and performing electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso-Discovery Company)) or non- Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL) -13) Generation. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein when the antibody binds to the plate, the anti-CD3 antibody that binds to the plate (E.g., 0.8μg/ml) in combination, when _{stimulated at 37°C and 5% CO 2} for e.g. 4 days, one or more cytokines (e.g., TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13), such as by, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)), where the concentration of anti-OX40 antibody is, for example, 0.7μg/ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg /ml and 50μg/ml, or between 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL- The production of 13) shows a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody (e.g., 0.8 μg/ml) bound on the plate and different concentrations (e.g., 0, 0.3, 1, 3, 6, 12, 25, and 50 μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) in the presence of the antibody of the present invention bound to the plate, for example, 37°C And culturing these PBMCs under 5% CO _{2 for} , for example, 4 days; and (b) Collect the supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery)) Or non-human primate (NHP) V-Plex assay kit (Meso Discovery Company)) to measure one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, Or IL-13).

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, when bound to the plate, in combination with the plate-bound anti-CD3 antibody (e.g., 0.8 μg/ml), induces e.g. PBMC or T after stimulation _{at e.g. 37°C and 5% CO 2 for e.g. 4 days} The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in the cell, such as by, for example, electrochemiluminescence (for example, human TH1/ TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measured by one or more cytokines (for example, TNFα , TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) are produced in, for example, 0.7μg/ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or a substantially increasing function of antibody concentration between 6.3μg/ml and 50μg/ml, as evaluated in an assay that includes, for example, the following steps: (a) Anti-CD3 antibody bound to the plate (e.g., 0.8μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml) In the presence of the antibody of the present invention bound to the plate _{, culture the PBMC at 37°C and 5% CO 2 for} , for example, 4 days; and (b) collect the supernatant and perform electrochemiluminescence (e.g., human TH1 /TH2 10-Plex Tissue Culture Kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (for example, TNFα, TNFβ) , IFNγ, GM-CSF, IL-2, IL-10, or IL-13). In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody, when bound to the plate, in combination with the plate-bound anti-CD3 antibody (e.g., 0.8 μg/ml), induces e.g. PBMC or T after stimulation _{at e.g. 37°C and 5% CO 2 for e.g. 4 days} The production of one or more cytokines (for example, TNFα, TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13) in the cell, such as by, for example, electrochemiluminescence (for example, human TH1/ TH2 10-Plex tissue culture kit (Meso Discovery Company) or non-human primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) is measured, where the concentration of anti-OX40 antibody is, for example, 0.7 μg/ ml and 50μg/ml, 1.6μg/ml and 50μg/ml, 3.1μg/ml and 50μg/ml, or between 6.3μg/ml and 50μg/ml, one or more cytokines (for example, TNFα, TNFβ, The production of IFNγ, GM-CSF, IL-2, IL-10, or IL-13) shows a sigmoidal dose-response curve, as evaluated in, for example, an assay that includes the following steps: (a) Anti-CD3 antibody bound to the plate ( For example, 0.8 μg/ml) and different concentrations (for example, 0, 0.3, 1, 3, 6, 12, 25, and 50 μg/ml; or 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ ml) plate-bound antibodies of the present invention are _{cultured at 37°C and 5% CO 2 for} , for example, 4 days; and (b) the supernatant is collected and subjected to electrochemiluminescence (e.g., Human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Company) or Non-Human Primate (NHP) V-Plex Assay Kit (Meso Discovery Company)) measures one or more cytokines (e.g., TNFα) , TNFβ, IFNγ, GM-CSF, IL-2, IL-10, or IL-13).

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of a VL domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is For example, a substantially increasing function of the antibody concentration between 0.2 μg/ml and 20 μg/ml, or between 2 μg/ml and 20 μg/ml, as evaluated in, for example, an assay including the following steps: (a) at 37°C, for example For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) for, for example, 7 minutes; (b) after sufficient washing, for example, 3 μg/ml is used for plate binding Anti-CD3 antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate-bound antibodies described herein, stimulate these _{at, for example, 37°C and 5% CO 2} cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with, for example anti-CD4 antibody and by means of flow cytometry by e.g. measuring low CFSE percentage of CD4 + cells to check CD4+ T cells proliferate. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein when the antibody is at a concentration of 20 μg/ml compared to at 2 μg/ In the presence of a concentration of ml, the antibody leads to a greater increase in CD4+ T cell proliferation, as assessed in, for example, an assay that includes the following steps: (a) For example, 10 μM carboxyfluorescein acetoacetate succinate at 37°C The amine ester (CFSE) labels, for example, the enriched CD4+ T cells for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02 , 0.2,2, and 20μg / ml), for example, plate-bound antibodies described herein, stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c) On, for example, day 4, perform cell staining with, for example, an anti-CD4 antibody and check for CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells by means of flow cytometry.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VL domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the concentration of the anti-OX40 antibody is For example, when 0.2μg/ml and 20μg/ml, or between 2μg/ml and 20μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve, as evaluated in, for example, an assay that includes the following steps: (a) In, for example, 37 For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at ℃ for, for example, 7 minutes; (b) After sufficient washing, for example, 3 μg/ml of for example Plate-bound anti-CD3 antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate-bound antibodies described herein, stimulate the _{plate at 37°C and 5% CO 2} and other cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody cell staining and by means of flow cytometry by e.g. measuring low CFSE CD4 + cell percentage to Check CD4+ T cell proliferation.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is For example, a substantially increasing function of the antibody concentration between 0.2 μg/ml and 20 μg/ml, or between 2 μg/ml and 20 μg/ml, as evaluated in, for example, an assay including the following steps: (a) at 37°C, for example For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) for, for example, 7 minutes; (b) After thorough washing, use, for example, 3 μg/ml, for example, a plate Bound anti-CD3 antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibodies such as those described herein, stimulate them _{at, for example, 37°C and 5% CO 2} cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with, for example anti-CD4 antibody and by means of flow cytometry by e.g. measuring low CFSE percentage of CD4 + cells to check CD4+ T cells proliferate. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein when the antibody is at a concentration of 20μg/ml compared to 2μg/ In the presence of a concentration of ml, the antibody leads to a greater increase in CD4+ T cell proliferation, as assessed in, for example, an assay that includes the following steps: (a) For example, 10 μM carboxyfluorescein acetoacetate succinate at 37°C The amine ester (CFSE) labels, for example, the enriched CD4+ T cells for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02 , 0.2,2, and 20μg / ml), for example, plate-bound antibodies described herein, stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c) On, for example, day 4, perform cell staining with, for example, an anti-CD4 antibody and check for CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells by means of flow cytometry.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include those with an amino acid sequence of at least 70% in the VH domain of pab1949 or pab2044 (e.g., SEQ ID NO: 16). %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity of the VH domain, wherein the antibody increases CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is For example, when 0.2μg/ml and 20μg/ml, or between 2μg/ml and 20μg/ml, CD4+ T cell proliferation shows an S-shaped dose response curve, as evaluated in, for example, an assay that includes the following steps: (a) In, for example, 37 For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at ℃ for, for example, 7 minutes; (b) After sufficient washing, for example, 3 μg/ml of for example Plate-bound anti-CD3 antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate-bound antibodies described herein, stimulate the _{plate at 37°C and 5% CO 2} and other cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody cell staining and by means of flow cytometry by e.g. measuring low CFSE CD4 + cell percentage to Check CD4+ T cell proliferation.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody increases CD4+ T cell proliferation, wherein the CD4+ T cell proliferation line is a substantially increasing function of the antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml, or 2 μg/ml and 20 μg/ml, as in For example, evaluation in an assay that includes the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidate (CFSE) at 37° C. for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound antibody such as described herein stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c), for example, on day 4, for example anti-CD4 antibody and the stained cells by flow Cytometry checks for CD4+ T cell proliferation by, for example, measuring the percentage of CFSE low CD4+ cells. In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, the antibody results in a greater increase in CD4+ T cell proliferation, as assessed in, for example, an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetyl succinimidyl acetate (CFSE) at 37° C. for, for example, 7 minutes; (b) After sufficient washing, for example, 3 μg/ml For example, plate-bound anti-CD3 antibodies and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as the plate-bound antibodies described herein, at 37° C. and 5% CO ₂ stimulating the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, for example anti-CD4 antibody and the stained cells by flow cytometry, for example, by measuring CFSE low CD4 + cells Percentage to check CD4+ T cell proliferation.

In certain embodiments, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) include: (i) and the amino acid of the VL domain of pab1949 or pab2044 (e.g., SEQ ID NO: 15) The sequence has a VL domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity; and (ii) with pab1949 or pab2044 (e.g., SEQ ID NO: 16) The amino acid sequence of the VH domain has a VH domain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity , Wherein the antibody increases the proliferation of CD4+ T cells, wherein when the anti-OX40 antibody concentration is between 0.2μg/ml and 20μg/ml, or 2μg/ml and 20μg/ml, the proliferation of CD4+ T cells shows an S-shaped dose response curve, As evaluated, for example, in an assay that includes the following steps: (a) For example, the enriched CD4+ T cells are labeled with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37° C., for example, 7 (B) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (e.g., 0.002, 0.02, 0.2, 2, and 20 μg/ml) of plate-bound as described herein antibody, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, cells were stained with anti-CD4 antibody and e.g. CD4+ T cell proliferation is checked by means of flow cytometry by, for example, measuring the percentage of CFSE low CD4+ cells.

In another aspect, provided herein are antibodies that bind to the same or overlapping epitopes of OX40 (e.g., epitopes of human OX40) as the antibodies described herein (e.g., pab1949 or pab2044). In some embodiments, the epitope of the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA analysis, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography mass spectrometry), array-based Oligopeptide scanning analysis, and/or mutagenic gene mapping (for example, site-directed mutagenesis gene mapping) is determined. For X-ray crystallography, any method known in the art can be used to complete the crystallization (For example, Giegé R et al. (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; Macpherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be studied using the well-known X-ray diffraction technique and computer software (such as X-PLOR (Yale University), 1992, refined by Molecular Simulations, Inc.); See, for example, Meth Enzymol (1985) Volumes 114 and 115, edited by Wyckoff HW et al.; U.S. Patent Application No. 2004/0014194), and BUSTER (Bricogne) G (1993) Acta Crystallography Series D: Biocrystallography 49 (Pt 1): 37-60; Brikonai G (1997) Methods of Enzymology 276A: 361-423, edited by Carter CW; Luo Weixi ( Roversi)P et al. (2000) Acta Crystallography Series D: Biological Crystallography 56(Pt 10): 1316-1323). Mapping of mutagenic genes can be done using any method known to those skilled in the art. See also For example, the description of the above-mentioned Champe M et al. (1995) and the above-mentioned Cunningham BC and Wells JA (1989) on mutagenesis techniques, including alanine partition mutagenesis techniques In a specific embodiment, alanine scanning mutagenesis studies are used to determine the epitope of the antibody. In addition, antibodies that recognize and bind to the same or overlapping epitopes of OX40 (e.g., human OX40) can use conventional techniques (e.g., immunoassays). Identification is performed, for example, by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen (i.e., competitive binding analysis). Competitive binding identification methods can also be used to determine whether two antibodies have similar binding specificities for epitopes. Competitive binding can be determined in an analysis, where the immunoglobulin under test inhibits the specific binding of the reference antibody to a common antigen (such as OX40). Many types of competitive binding analysis are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunoassay (EIA), sandwich competition analysis (see Stahli C, etc.) Human, (1983) Methods Enzymol (Methods Enzymol) 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland (Kirkland) TN et al., (1986) Journal of Immunology 137:3614- 9); solid-phase direct labeling analysis, solid-phase direct labeling sandwich analysis (see Harlow E and Rane D, (1988) Antibody: Laboratory Manual, Cold Spring Harbor Laboratory Press); solid phase with I-125 label Direct labeling of RIA (see Morel GA et al. (1988) Mol Immunol 25(1): 7-15); solid-phase direct biotin-avidin EIA (Cheung ) RC et al. (1990) Virology 176:546-52); and direct labeling of RIA (Moldenhauer G et al. (1990) Scandinavian Journal of Immunology ( Scand J Immunol) 32: 77-82). Typically, such analysis involves the use of purified antigens (e.g., OX40 (such as human OX40)) that bind to a solid surface or cell with either of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. It can be tested in the presence of immunoglobulin by determining the amount of label bound to a solid surface or cell Instead, measure competitive inhibition. Usually, the test immunoglobulin is present in excess. Generally, when the competing antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75% Or more. The competitive binding identification method can be configured in many different formats using labeled antigen or labeled antibody. In a common version of this analysis, the antigen is immobilized on a 96-well plate. Then, radioactivity or enzyme tags are used to measure the ability of the unlabeled antibody to block the labeled antibody from binding to the antigen. For further details, see, for example, Wagner C, et al., (1983) Journal of Immunology 130: 2308-2315; Wagner C, et al., (1984) Journal of Immunological Methods 68: 269-274; Kuroki ( Kuroki M et al. (1990) Cancer Research 50: 4872-4879; Kuroki M et al. (1992) Immunol Invest 21: 523-538; Kuroki M et al. (1992) Fusion tumor (Hybridoma) 11: 391-407 and Antibodies: Laboratory Manual, edited by Harlow E and Rane D above, pp. 386-389.

In one embodiment, surface plasmon resonance (BIAcore®) is used, for example by the'in tandem approach' (such as Abdiche YN et al., (2009) Analytical Biochem ) 386: 172-180) to conduct a competition analysis, thereby immobilizing the OX40 antigen on the surface of the wafer (for example, a CM5 sensor wafer), and then allowing the anti-OX40 antibody to run over the wafer. To determine whether an antibody competes with the anti-OX40 antibody described herein, the anti-OX40 antibody is first run over the surface of the wafer to reach saturation, and then the potential competing antibody is added. Compared to non-competitive controls, The binding of competing antibodies can then be determined and quantified.

In certain aspects, competitive binding analysis can be used to determine whether an antibody system is competitively blocked by another antibody, for example, in a dose-dependent manner, for example, when two antibodies are identified in a competitive binding analysis (such as a competitive ELISA analysis). In the case of the same or spatially overlapping epitopes, the antibody and the reference antibody bind to substantially the same epitope or overlapping epitopes, and the analysis can be configured in many different forms using labeled antigens or labeled antibodies. In a specific embodiment, in a competitive binding analysis, the antibody may interact with the antibody described herein (e.g., antibody pab1949 or pab2044), or its chimeric or Fab antibody, or include the antibody described herein (e.g., pab1949 or pab2044). ) VH CDR and VL CDR antibodies are tested.

In another aspect, provided herein is an antibody that competes (for example, in a dose-dependent manner) with the antibody described herein (for example, pab1949 or pab2044) for binding to OX40 (for example, human OX40), such as those skilled in the art As determined by known or described analysis (e.g., ELISA competitive analysis or surface plasmon resonance). In another aspect, provided herein is an antibody that competitively inhibits (e.g., in a dose-dependent manner) an antibody (e.g., pab1949 or pab2044) described herein that binds to OX40 (e.g., human OX40), if familiar with the technology Known or determined by analysis described herein (e.g., ELISA competitive analysis, or suspension array or surface plasmon resonance analysis). In specific embodiments, such competitive blocking antibody activates, induces or enhances one or more OX40 activities. In specific aspects, provided herein are antibodies that include the amino acid sequences described herein (for example, antibodies pab1949 or pab2044). VL and/or VH amino acid sequences) compete (e.g., in a dose-dependent manner) for antibodies that specifically bind to OX40 (e.g., human OX40), as in the analysis known to those skilled in the art or described herein (e.g., , ELISA competitive analysis, or suspension array or surface plasmon resonance analysis) determined.

In certain embodiments, provided herein are antibodies that compete with the antibodies described herein for binding to OX40 (e.g., human OX40) to the same extent as the antibodies described herein that compete for binding to OX40 (e.g., human OX40) to the same extent . In some embodiments, there is provided a first antibody that competes with the antibody described herein for binding to OX40 (e.g., human OX40), wherein the first antibody competes for binding in an analysis, and the analysis includes the following steps: (a) Culturing OX40 transfected cells in a container with the first antibody in an unlabeled form; and (b) adding the antibody described herein in a labeled form to the container and culturing the cells in the container; and (c) detecting the binding of the cells The antibodies described herein in labeled form. In certain embodiments, provided herein is a first antibody that competes with an antibody described herein for binding to OX40 (e.g., human OX40), wherein the competition is presented as a reduction in the binding of the first antibody to OX40 by more than 80% (e.g., 85%, 90%, 95% or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85% to 95%).

In specific aspects, provided herein are those that include the VL domain having the amino acid sequence listed in SEQ ID NO: 15 and the VH domain having the amino acid sequence listed in SEQ ID NO: 16 Antibodies compete (e.g., in a dose-dependent manner) to specifically bind to OX40 (e.g. For example, human OX40) antibodies.

In specific aspects, provided herein and including (i) including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequence of the VL CDR listed in Table 1; and (ii) including Antibodies of the VH CDR1, VH CDR2, and VH domain of the VH CDR3 of the amino acid sequence of the CDRs in Table 2 compete (e.g., in a dose-dependent manner) for antibodies that specifically bind to OX40 (e.g., human OX40).

In a specific embodiment, the anti-system described herein includes a VL domain having the amino acid sequence set forth in SEQ ID NO: 15 and a VL domain having the amino acid sequence set forth in SEQ ID NO: 16 The VH domain of the antibody competitively blocks (e.g., in a dose-dependent manner) an antibody that specifically binds to OX40 (e.g., human OX40).

In another specific embodiment, the anti-system described herein includes (i) VL domains including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the CDRs listed in Table 1; and (ii ) An antibody that includes antibodies competitively blocking (for example, in a dose-dependent manner) of the VH domains of VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 2.

In a specific aspect, an antibody that immunospecifically binds to the same epitope as an antibody of pab1949 or pab2044 is provided herein to specifically bind to OX40 (for example, human OX40). Those familiar with the technique known or described herein (for example, X-ray crystallography, coupled mass spectrometry (for example, liquid chromatography mass spectrometry) hydrogen/deuterium exchange, alanine scanning, ELISA Analysis etc.) can be used to determine whether two antibodies bind to the same epitope.

In a specific embodiment, the antibodies described herein immunospecifically bind to the same epitope bound by pab1949 or pab2044 or an epitope that overlaps the epitope.

In another specific embodiment, an antibody described herein and a VL domain comprising (i) VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of the CDRs listed in Table 1; and (ii) Antibodies including the VH CDR1, VH CDR2, and VH CDR3 VH domains having the amino acid sequences of the CDRs listed in Table 2 immunospecifically bind to the same epitope.

In a specific aspect, the binding between the antibody described herein and the variant OX40 is substantially weaker relative to the binding between the antibody and the human OX40 sequence of SEQ ID NO: 55, and wherein the variant OX40 includes SEQ ID The sequence of NO: 55, except for one amino acid mutation (for example, substitution) selected from the group consisting of N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof. In some embodiments, the variant OX40 includes the sequence of SEQ ID NO: 55, except for any one mutation selected from the group consisting of: N60A, R62A, R80A, L88A, P93A, P99A, and P115A . In some embodiments, the variant OX40 includes the sequence of SEQ ID NO: 55, except for any two, three, four, five, six, or seven mutations selected from the group consisting of: W58A, N60A , R62A, R80A, L88A, P93A, P99A, and P115A. In some embodiments, the variant OX40 includes the sequence of SEQ ID NO: 55, except for the amine group Acid mutations W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A.

In a specific aspect, the antibody described herein specifically binds to an epitope of the human OX40 sequence, which epitope includes, consists essentially of, or consists of residues selected from the group consisting of SEQ ID NO: 55, the The group consists of the following: 60, 62, 80, 88, 93, 99, 115, and combinations thereof. In some embodiments, the epitope includes, consists essentially of, or consists of any one residue selected from the group consisting of: 60, 62, 80 of SEQ ID NO: 55 , 88, 93, 99, and 115. In some embodiments, the epitope includes, consists essentially of, or consists of any two, three, four, five, six, or seven residues selected from the group consisting of: 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO:55. In some embodiments, the epitope includes, consists essentially of, or consists of residues 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO: 55.

In a specific embodiment, the antibody described herein specifically binds to an epitope of SEQ ID NO: 55, which epitope includes, consists essentially of, or consists of a residue selected from the group consisting of The following items are composed: 60, 62, 80, 88, 93, 99, 115, and combinations thereof. In some embodiments, the epitope includes, consists essentially of, or consists of any one residue selected from the group consisting of: 60, 62, 80 of SEQ ID NO: 55 , 88, 93, 99, and 115. In some embodiments, the epitope includes, consists essentially of, or consists of any two, three, four, five, six, or seven residues selected from the group consisting of It consists of the following: 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO: 55. In some embodiments, the epitope includes, consists essentially of, or consists of residues 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO: 55.

In a specific aspect, the antibody described herein specifically binds to at least one residue of SEQ ID NO: 55 selected from the group consisting of: 60, 62, 80, 88, 93, 99, 115 , And its combination. In some embodiments, the antibodies described herein specifically bind to any one residue, or any two, three, four, five, six, or seven residues selected from the group consisting of: SEQ ID NO: 55 of 60, 62, 80, 88, 93, 99, and 115. In some embodiments, the antibodies described herein specifically bind to any two, three, four, five, six, or seven residues selected from the group consisting of: 58 of SEQ ID NO: 55 , 60, 62, 80, 88, 93, 99, and 115. In some embodiments, the antibodies described herein specifically bind to residues 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO:55.

In a specific aspect, compared to the human OX40 sequence that binds to SEQ ID NO: 55, the antibody described herein displays the same protein as SEQ ID NO: 55 (except for the presence of an amino acid mutation selected from the group (e.g., , Substituted), the group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof) reduced binding or non-binding. In some embodiments, the protein is the same as SEQ ID NO: 55, except that there are amino acid mutations including any one mutation selected from the following group, or any two, three, four, five, six, or seven mutations. Change, the group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, and P115A. In some embodiments, the protein is the same as SEQ ID NO: 55, except that there are amino acid mutations including any two, three, four, five, six, or seven mutations selected from the group consisting of Composition: W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A. In some embodiments, the protein is the same as SEQ ID NO: 55, except that there are amino acid substitutions including mutations W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A.

In certain embodiments, the epitopes of the antibodies described herein are used as immunogens to produce antibodies. For methods used to produce antibodies, see, for example, section 5.3 below.

In specific aspects, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) function as agonists.

In certain embodiments, relative to the activity of OX40 (e.g., human OX40) in the absence or presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), immunospecific binding to OX40 (E.g., human OX40) antibodies described herein increase the activity of OX40 (e.g., human OX40) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4 Times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as determined by methods described herein and/or known to those skilled in the art. In some embodiments, relative to The activity of OX40 (for example, human OX40) in the absence of any antibody or an unrelated antibody (for example, an antibody that does not immunospecifically bind to OX40), which immunospecifically binds to OX40 (for example, human OX40) is described here The antibody increased the activity of OX40 (e.g., human OX40) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as determined by methods described herein and/or known to those skilled in the art. Non-limiting examples of OX40 (e.g., human OX40) activity can include OX40 (e.g., human OX40) signaling, cell proliferation, cell survival, and cytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13). In certain embodiments, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) induce, enhance, or increase OX40 (e.g., human OX40) activity. In a specific embodiment, the increase in OX40 activity is determined as described in the examples below.

In certain aspects, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) induce, enhance, or increase cell proliferation of cells that express OX40 and respond to OX40 signaling (e.g., in response to OX40 stimulation and OX40 Cells (such as T cells) that proliferate for communication). The cell proliferation assay is as described in the art (for example, ³ H-thymidine incorporation method, BrdU incorporation method, or CFSE method, as described in Example 2), and can be easily performed by those skilled in the art Implement. In a specific embodiment, compared to using only T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex stimulating agent (Such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells, using T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) , Or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) in immunospecific binding to OX40 (such as human OX40) described here The presence of antibodies has increased cell proliferation.

In a specific embodiment, relative to the stimulation of OX40 (e.g., human OX40) activity in the absence of any antibody or the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to OX40), immunospecific binding of OX40 (E.g., human OX40) antibodies described herein increase cell proliferation (e.g., T cells (such as CD4 and CD8 effector T cells)) at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold Times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70-fold, 80-fold, 90-fold, or 100-fold, as described herein or by methods known to those skilled in the art (for example, ³ H-thymidine incorporation method, BrdU incorporation method, or CFSE method, Measured as described in Example 2 below). In a specific embodiment, relative to the activity of OX40 (e.g., human OX40) in the absence of any antibody or the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to OX40), immunospecific binding to OX40 ( For example, human OX40) antibodies described herein increase cell proliferation (eg, T cells (such as CD4 and CD8 effector T cells)) by at least about 5%, 10%, 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, if This describes or is determined by methods known to those skilled in the art (for example, ³ H-thymidine incorporation method, BrdU incorporation method, or CFSE method, as described in Example 2 below). In a specific embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) increases CD4+ T cell proliferation, where the CD4+ T cell proliferation line is at an antibody concentration between, for example, 0.2 μg/ml and 20 μg/ml The substantially increasing function of, as evaluated in, for example, an assay comprising the following steps: (a) for example enriched with 10 μM carboxy fluorescein acetoacetate succinimidyl ester (CFSE) at 37°C, for example CD4+ T cells are labeled for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) for example, plate-bound antibody described herein, for example, in the case of 37 [deg.] C and 5% CO ₂ to stimulate the cells (e.g., in the well ¹⁰⁵ cells); and (c), for example, on day 4, with For example, anti-CD4 antibody is used for cell staining and CD4+ T cell proliferation is checked by means of flow cytometry by, for example, measuring the percentage of CFSE low CD4+ cells. In a specific embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) increases CD4+ T cell proliferation, where the CD4+ T cell proliferation line is at an antibody concentration between, for example, 2 μg/ml and 20 μg/ml A substantially incremental function, as evaluated in, for example, an assay that includes the following steps: (a) For example, the concentration of CD4+ is enriched with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C. T cells are labeled for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) for example, plate-bound antibodies described herein, stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c), for example, on day 4, with e.g. The cells are stained with anti-CD4 antibodies and CD4+ T cell proliferation is checked by means of flow cytometry by, for example, measuring the percentage of CFSE low CD4+ cells. In a specific embodiment, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) increase CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 0.2 μg/ml and 20 μg/ml, CD4+ T cell proliferation exhibits a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) For example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) is used for e.g. enrichment at 37°C. The collected CD4+ T cells are labeled for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ ml), for example, plate-bound antibody described herein, for example, 37 [deg.] C and 5% CO stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case _2; and (c) at day 4 e.g. Cells are stained with, for example, anti-CD4 antibodies and CD4+ T cell proliferation is checked by, for example, measuring the percentage of CFSE low CD4+ cells by means of flow cytometry. In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) increase CD4+ T cell proliferation, wherein when the anti-OX40 antibody concentration is between, for example, 2 μg/ml and 20 μg/ml, CD4+ T Cell proliferation shows a sigmoidal dose response curve, as evaluated in an assay that includes, for example, the following steps: (a) for example enrichment with, for example, 10 μM carboxyfluorescein acetoacetate succinimidyl ester (CFSE) at 37°C Labeling of CD4+ T cells for, for example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml ), for example, plate-bound antibody described herein, for example, 37 [deg.] C and 5% CO stimulating the cells (e.g., in the well ¹⁰⁵ cells) in the case _2; and (c), for example, on day 4, Cells are stained with, for example, an anti-CD4 antibody and CD4+ T cell proliferation is checked by means of flow cytometry by, for example, measuring the percentage of CFSE low CD4+ cells. In a specific embodiment, when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 2 μg/ml, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) results in a more proliferation of CD4+ T cells. A large increase, as evaluated in, for example, an assay comprising the following steps: (a) Labeling, for example, enriched CD4+ T cells with, for example, 10 μM carboxyfluorescein acetoacetate succinimidate (CFSE) at 37°C For example, 7 minutes; (b) After sufficient washing, use, for example, 3 μg/ml of plate-bound anti-CD3 antibody and different concentrations (for example, 0.002, 0.02, 0.2, 2, and 20 μg/ml) such as described herein plate-bound antibodies which stimulate the cells (e.g., in the well ¹⁰⁵ cells) in the case of, for example, 37 [deg.] C and 5% CO _2; and (c), for example, on day 4, with the anti-CD4 antibody e.g. Cell staining is performed and CD4+ T cell proliferation is checked by means of flow cytometry by, for example, measuring the percentage of CFSE low CD4+ cells.

In some embodiments, T cells stimulated with T cell mitogens (e.g., anti-CD3 antibodies or phorbol esters) (e.g., CD4 ⁺ or CD8 ⁺ effect T cells) in the presence of antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) have an increase of at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold , 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 Fold, 90-fold, or 100-fold cell proliferation, as described herein or by methods known to those skilled in the art (for example, ³ H-thymidine incorporation method, BrdU incorporation method, or CFSE method, such as Described in Example 2 below) determined. In some embodiments, compared to stimulating the antibody with only T cell mitogens or T cell receptor complex stimulators, such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex (Such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells, using T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) , Or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) in immunospecific binding to OX40 (such as human OX40) described here In the presence of the antibody, there is an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of cell proliferation, as described herein or by methods known to those skilled in the art (for example, ³ H-thymus Pyrimidine nucleoside incorporation method, BrdU incorporation method or CFSE method, as described in Example 2 below). In a specific embodiment, cell proliferation is measured as described in Example 2 below. In a specific embodiment, 5 μg/ml of the OX40 antibody described herein increases the proliferation of human CD4 T cells treated with 3 μg/ml anti-CD3 antibody by at least 20%. In a specific embodiment, 5 μg/ml of the OX40 antibody described herein increases the proliferation of human CD4 T cells treated with 3 μg/ml anti-CD3 antibody by at least 30%. In a specific embodiment, 5 μg/ml of the OX40 antibody described herein increases the proliferation of human CD4 T cells treated with 3 μg/ml anti-CD3 antibody by at least 40%. In a specific embodiment, 5 μg/ml of the OX40 antibody described herein increases the proliferation of human CD4 T cells treated with 3 μg/ml anti-CD3 antibody by at least 50%.

In certain aspects, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) increase the survival of cells (e.g., T cells (e.g., CD4 and CD8 effector T cells)). In a specific embodiment, compared to T cells stimulated with only T cell mitogens, T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol) are used. Ethyl ester (PMA), or TCR complex stimulating antibody (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (for example, CD4 ⁺ or CD8 ⁺ effector T cells) in the immune specific binding OX40 (for example, human OX40 ) Has increased survival in the presence of the antibodies described herein. The cell survival assay is as described in the art (for example, trypan blue exclusion assay) and can be easily performed by a person familiar with the technique.

In a specific embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) increases cell survival (e.g., T cells (such as CD4 and CD8 effector T cells)) by at least about 1.2-fold, 1.3-fold , 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 Times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, as described herein or by a method known to those skilled in the art (for example, trypan blue exclusion assay), Measured in the absence of any antibodies or irrelevant antibodies (for example, antibodies that do not immunospecifically bind to OX40). In a specific embodiment, relative to the activity of OX40 (e.g., human OX40) in the absence of any antibody or the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to OX40), immunospecific binding to OX40 ( For example, human OX40) antibodies described herein increase cell survival (e.g., T cells (such as CD4 and CD8 effector T cells)) by at least about 5%, 10%, 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, if This describes or is determined by a method known to those skilled in the art (for example, trypan blue exclusion assay).

In some embodiments, compared to only stimulating T cell mitogens or T cell receptor complex stimulators, such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex stimulating antibody (Such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells, T cells stimulated with T cell mitogens (for example, anti-CD3 antibodies or phorbol esters) (for example, CD4 ⁺ or CD8 ⁺ effector T cells) In the presence of antibodies described herein that immunospecifically bind to OX40 (for example, human OX40), there is an increase of at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times Or 100-fold cell survival, as determined by methods described herein or known to those skilled in the art (for example, trypan blue exclusion assay). In some embodiments, compared to T cells stimulated with only T cell mitogens, T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol beta) are used. Ester (PMA), or TCR complex stimulating antibodies (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (for example, CD4 ⁺ or CD8 ⁺ effector T cells) specifically bind to OX40 (for example, human OX40) In the presence of the antibodies described herein, there is an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of cell survival, as described herein or methods known to those skilled in the art (e.g., Trypan blue exclusion assay).

In certain embodiments, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) prevent effector T cells (e.g., CD4 ⁺ and CD8 ⁺ effector T cells) from priming-induced cell death.

In a specific embodiment, compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), in the presence or absence of OX40 (e.g., human OX40) stimulation Cytokines are produced, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) produce cytokines (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, And/or IL-13) induce, enhance, or increase at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as described here (see the following examples, such as example 2) or those who are familiar with the technology Measured by known methods. In a specific embodiment, compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), in the presence or absence of OX40 (e.g., human OX40) stimulation Cytokines are produced, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) produce cytokines (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, And/or IL-13) induce or increase at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times Times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as described here (see below Examples, as measured in Example 2) or methods known to those skilled in the art.

In certain embodiments, compared to only stimulating T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex stimulation T cells stimulated by antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies), use T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) ), or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells (for example, CD4 ⁺ or CD8 ⁺ effector T cells) that immunospecifically bind to OX40 (for example, human OX40). In the presence of the described antibody, there is an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of cytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL- 10. and/or IL-13), as determined by methods described herein or known to those skilled in the art (e.g., ELISA assay or as described in the examples below). In some embodiments, compared to stimulating the antibody with only T cell mitogens or T cell receptor complex stimulators, such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex (Such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells, using T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) , Or TCR complex stimulating antibody (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) in the immune specific binding OX40 (such as human OX40) described here In the presence of the antibody, it has an increase of at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold cytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13), as described herein or known to those skilled in the art (e.g., ELISA assay or as in the following examples Said) measured.

In a specific embodiment, relative to IL-2 production in the absence of any antibody or the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to OX40), immunospecific binding to OX40 (e.g., human OX40 ) The antibodies described herein increase IL-2 production in response to staphylococcal enterotoxin A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold Times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as determined by the method described here (see the following example, such as example 2) or by a method known to those skilled in the art.

In certain embodiments, T cells stimulated with staphylococcal enterotoxin A (SEA) (for example, CD4 ⁺ or CD8 ⁺ T cells) specifically bind to OX40 ( For example, human OX40) has an increase of at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times IL- 2 Produced, as determined by methods described herein or known to those skilled in the art (for example, ELISA assay or as described in the examples below).

In a specific embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) is combined with Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml) when, for example, 37°C, Stimulation under 5% CO ₂ and 97% humidity for example 5 days later induces the production of IL-2 in PBMC, such as by electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (microscopic findings) Company)) measured in which the production of IL-2 is a substantially increasing function of the antibody concentration between, for example, 0.032 μg/ml and 20 μg/ml. In some embodiments, the IL-2 production induced by the binding of the antibody and SEA is, for example, 0.16 μg/ml and 20 μg/ml, 0.8 μg/ml and 20 μg/ml, 4 μg/ml and 20 μg/ml, 0.032 μg /ml and 4 μg/ml, 0.16 μg/ml and 4 μg/ml, or 0.8 μg/ml and 4 μg/ml as a substantially increasing function of antibody concentration. In another embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40), in combination with Staphylococcal Enterotoxin A (SEA), induces, for example, the production of IL-2 in PBMC, where IL The production of -2 is a substantially increasing function of the antibody concentration between, for example, 0.032 μg/ml and 20 μg/ml, as assessed in, for example, an assay that includes the following steps: (a) in the absence or presence of different concentrations The antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100 ng/ml SEA, _{cultured at, for example, 37° C., 5% CO 2} , and 97% humidity such PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. electrochemiluminescence (e.g., a human TH1 / TH2 10-Plex tissue culture kit ( Meso-discovered company)) Measure the titer of IL-2. In certain embodiments, antibodies described herein that immunospecifically bind to OX40 (eg, human OX40), in combination with Staphylococcal Enterotoxin A (SEA), induce, for example, the production of IL-2 in PBMC, where IL The production line of -2 is, for example, 0.16μg/ml and 20μg/ml, 0.8μg/ml and 20μg/ml, 4μg/ml and 20μg/ml, 0.032μg/ml and 4μg/ml, 0.16μg/ml and 4μg/ml , Or a substantially increasing function of the antibody concentration between 0.8 μg/ml and 4 μg/ml, as evaluated in an assay including, for example, the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100 ng/ml SEA, at 37°C, 5% CO ₂ , and 97% humidity under such PBMC (e.g. , in the hole ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. electrochemiluminescence (e.g., a human TH1 / TH2 10-Plex tissue culture kit (meso found Corporation) ) Measure the titer of IL-2. In a specific embodiment, when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 0.032 μg/ml, when the _{stimulation is continued at, for example, 37° C., 5% CO 2} , and 97% humidity for, for example, 5 days, the immune system The antibodies described herein that specifically bind to OX40 (e.g., human OX40) result in more IL-2 production in response to Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml). In a specific embodiment, when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 0.16 μg/ml, after stimulation at 37° C., 5% CO ₂ , and 97% humidity for, for example, 5 days, The antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) in response to Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml) result in more IL-2 production.

In a specific embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) is combined with Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml) when, for example, 37°C, Stimulation under 5% CO ₂ and 97% humidity for example 5 days later induces the production of IL-2 in PBMC, such as by electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (microscopic findings) Company)) measured, wherein when the anti-OX40 antibody concentration is between, for example, 0.032 μg/ml and 20 μg/ml, the production of IL-2 shows an S-shaped dose response curve. In some embodiments, when the concentration of anti-OX40 antibody is between 0.16 μg/ml and 20 μg/ml, 0.8 μg/ml and 20 μg/ml, 4 μg/ml and 20 μg/ml, 0.032 μg/ml and 4 μg/ml, 0.16 μg/ml and 20 μg/ml, for example, When between μg/ml and 4μg/ml, or between 0.8μg/ml and 4μg/ml, the IL-2 production induced by the combination of antibody and SEA showed a sigmoidal dose response curve. In another embodiment, an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40), in combination with Staphylococcal Enterotoxin A (SEA), induces, for example, the production of IL-2 in PBMC, where When the anti-OX40 antibody concentration is between, for example, 0.032 μg/ml and 20 μg/ml, IL-2 production shows a sigmoidal dose response curve, as assessed in, for example, an assay that includes the following steps: (a) In the absence or presence of different concentrations The antibody (e.g., 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg/ml) and, for example, 100 ng/ml SEA, at 37° C., 5% CO ₂ , and 97% humidity such culturing PBMC (e.g., in the well ¹⁰⁵ cells) for five days, for example; and (b) the supernatant was collected and clarified by e.g. electrochemiluminescence (e.g., a human TH1 / TH2 10-Plex tissue culture kit (Meso-Discovery Company)) Measure the titer of IL-2. In certain embodiments, antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40), in combination with Staphylococcal Enterotoxin A (SEA), induce, for example, the production of IL-2 in PBMC, where The concentration of anti-OX40 antibody is, for example, 0.16 μg/ml and 20 μg/ml, 0.8 μg/ml and 20 μg/ml, 4 μg/ml and 20 μg/ml, 0.032 μg/ml and 4 μg/ml, 0.16 μg/ml and 4 μg/ml, Or between 0.8 μg/ml and 4 μg/ml, IL-2 production shows a sigmoidal dose response curve, as evaluated in an assay that includes the following steps: (a) in the absence or presence of different concentrations of the antibody (e.g. , 20,4,0.8,0.16,0.032,0.0064,0.00128, and 0.000256μg / ml) and for example SEA 100ng / ml and incubated at e.g. 37 ℃, 5% CO _2, 97% humidity, and these PBMC ( For example, 10 ⁵ cells in the well) last for example 5 days; and (b) collect the clear supernatant and use, for example, electrochemiluminescence (e.g., human TH1/TH2 10-Plex tissue culture kit (Meso Discovery Inc.) )) Measure the titer of IL-2. In a specific embodiment, when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 0.032 μg/ml, when the _{stimulation is continued at, for example, 37° C., 5% CO 2} , and 97% humidity for, for example, 5 days, the immune system The antibodies described herein that specifically bind to OX40 (e.g., human OX40) result in more IL-2 production in response to Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml). In a specific embodiment, when the antibody is present at a concentration of 20 μg/ml compared to a concentration of 0.16 μg/ml, after stimulation at 37° C., 5% CO ₂ , and 97% humidity for, for example, 5 days, The antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) respond to Staphylococcal Enterotoxin A (SEA) (e.g., 100 ng/ml) resulting in more IL-2 production.

In a specific embodiment, relative to IL-10 production in the absence of any antibodies or the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), immunospecific binding to OX40 (e.g., human OX40 ) The antibody described herein reduces IL-10 production in response to staphylococcal enterotoxin A (SEA) stimulation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% %, 45%, or 50%, as determined by the methods described herein (see the examples below, such as Example 2) or methods known to those skilled in the art.

In certain embodiments, T cells stimulated with staphylococcal enterotoxin A (SEA) (for example, CD4 ⁺ or CD8 ⁺ T cells) specifically bind to OX40 ( For example, human OX40) has a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the presence of the antibodies described herein. The production of IL-10 is as determined by methods described herein or known to those skilled in the art (e.g., ELISA assay or as described in the examples below).

In a specific embodiment, when bound to activated regulatory T In cells, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) bind to activated Fcγ receptors selected from the group consisting of CD16, CD32A, and CD64 to a degree greater than (e.g., 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times , 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) when bound to activated effector T cells, the antibody binds to an activated Fcγ receptor selected from the group consisting of CD16, CD32A, and CD64 The degree of body is determined by a method described herein or known to those skilled in the art (for example, the Fcγ receptor IIIA (CD16) reporter assay or as described in the following examples). In a specific embodiment, activated Fcγ receptors are expressed on cells selected from the group consisting of: myeloid-derived effector cells and lymphocyte-derived effector cells. In a specific embodiment, the Fcγ receptor system CD16 is activated.

In a specific embodiment, the antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) when bound to activated regulatory T cells cause an activated Fcγ receptor selected from the group consisting of CD16, CD32A, and CD64. The activation of the body is stronger than when binding to activated effector T cells, the antibody causes activation of an activated Fcγ receptor selected from the group consisting of CD16, CD32A, and CD64. In a specific embodiment, when an antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) binds to activated regulatory T cells, the activation of activated Fcγ receptors is stronger than when immunospecifically binds to OX40 ( For example, when the antibodies described herein of human OX40) bind to activated effector T cells, the activation of Fcγ receptors is activated. Change at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times , 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as described herein or by methods known to those familiar with the art (for example, Fcγ receptor IIIA (CD16) report assay or as described in the examples below). In a specific embodiment, activated Fcγ receptors are expressed on cells selected from the group consisting of: myeloid-derived effector cells and lymphocyte-derived effector cells. In a specific embodiment, the Fcγ receptor system CD16 is activated.

In a specific aspect, provided herein is an antagonist antibody that immunospecifically binds to OX40 (e.g., human OX40).

The activation of OX40 signaling depends on receptor clustering to form a more ordered receptor complex that effectively recruits the top adaptor protein to drive intracellular signal transduction. Without being bound by theory, anti-OX40 agonist antibodies can be used via bivalent antibody arms (ie, two antibody arms each binding to OX40 antigen) and/or via Fc-Fc receptors (FcR) co-joined to helper bone marrow or lymphoid cells. ) To mediate receptor clustering. Therefore, one approach for the development of anti-OX40 antagonist antibodies is to select antibodies that compete with the OX40 ligand (OX40L) for binding to OX40, reduce or eliminate the binding of the Fc region of the antibody to the Fc receptor, and/or adopt a monovalent antibody format . The monovalent antibody format may include structurally monovalent antibodies, such as, but not limited to, an anti-OX40 antibody that includes only one antigen-binding domain (e.g., only one Fab arm), or only one that binds to OX40 (e.g., human OX40). Chain pairing or antigen binding paired with a fragment of the heavy chain (for example, an Fc fragment) Binding domain antibodies. The monovalent antibody form may also include a functionally monovalent antibody, for example, an antibody that includes only one antigen-binding domain that binds to OX40 (e.g., human OX40) that is paired with a second antigen-binding domain that does not bind to an antigen expressed by human immune cells ( That is, the antibody includes two antigen-binding domains, but only one antigen-binding domain binds to OX40).

The examples of mutations in the Fc region of the IgG constant domain discussed above can reduce Fc receptor binding or can remove potential glycosylation sites. In certain embodiments, the heavy chain constant region of an antibody that immunospecifically binds to OX40 (e.g., human OX40) as described herein includes a mutation selected from the group consisting of: N297A, N297Q, D265A, C127S, S228P, and combinations thereof. In some embodiments, the mutation is N297A, N297Q, D265A, or a combination thereof. In some embodiments, the mutation is C127S. In some embodiments, the mutation is S228P. In one embodiment, the heavy chain constant region of an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes a mutation selected from the group consisting of D265A, P329A, and Its combination. In certain embodiments, the heavy chain constant region is selected from the group consisting of immunoglobulins IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In certain embodiments, the immunoglobulin is a human immunoglobulin. Human immunoglobulins containing mutations (e.g., substitutions) are also referred to herein as human immunoglobulins. In a specific aspect, an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes an immunoglobulin IgG ₁ heavy chain constant region, wherein _{the amino acid sequence of the IgG 1} heavy chain constant region includes the group selected from Group of mutations, the group consists of the following: N297A, N297Q, D265A, or a combination thereof. In one aspect, an antibody that immunospecifically binds to OX40 (e.g., human OX40) as described herein includes an immunoglobulin IgG ₁ heavy chain constant region, wherein _{the amino acid sequence of the IgG 1} heavy chain constant region is selected from the group consisting of The group consists of the following: D265A, P329A, and combinations thereof. In a specific aspect, an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes an immunoglobulin IgG ₂ heavy chain constant region, wherein _{the amino acid sequence of the IgG 2} heavy chain constant region includes a C127S mutation. In a specific aspect, an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes an immunoglobulin IgG ₄ heavy chain constant region, wherein _{the amino acid sequence of the IgG 4} heavy chain constant region includes the S228P mutation. In certain embodiments, the resistance system is antagonistic.

In a specific aspect, an antibody that immunospecifically binds to OX40 (e.g., human OX40) as described herein is selected from the group consisting of Fab, Fab', F(ab') ₂ , and scFv fragments, wherein Fab, Fab', F(ab') ₂ , or scFv fragments include the heavy chain variable region sequence and light chain variable region sequence of an anti-OX40 antigen binding domain or antibody as described herein. _{Fab, Fab', F(ab') 2} , or scFv fragments can be produced by any technique known to those skilled in the art (including but not limited to those discussed in Section 5.3 below). In some embodiments, the Fab, Fab', F(ab') ₂ , or scFv fragment further includes a portion that extends the half-life of the antibody in vivo. This part is also called the "half-life extension part". Any part known to those skilled in the art for extending the _{half-life of Fab, Fab', F(ab') 2} , or scFv fragments in vivo can be used. For example, the half-life extending portion may include an Fc region, polymer, albumin, or albumin binding protein or compound. The polymer may include natural or synthetic, optionally substituted linear or branched polyalkylene, polyalkylene, polyoxyalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxy Base polyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxyl, methyl, or methoxy. _{In some embodiments, Fab, Fab', F(ab') 2} , or scFv fragments can be modified by adding one or more C-terminal amino acids for attaching half-life extension moieties. In some embodiments, the half-life extension portion is polyethylene glycol or human serum albumin. In some embodiments, Fab, Fab', F(ab') ₂ , or scFv fragments are fused to the Fc region. In certain embodiments, the resistance system is antagonistic.

In a specific aspect, an antibody that immunospecifically binds to OX40 (e.g., human OX40) includes a heavy chain and a light chain (that is, the antibody does not include any other heavy or light chains, and includes a single heavy chain-light chain pair, It consists essentially of, or consists of, the heavy chain and the light chain, respectively, including the heavy chain variable region sequence and the light chain variable region sequence of the anti-OX40 antigen-binding domain or antibody as described herein. In certain embodiments, the heavy chain includes mutations selected from the group consisting of N297A, N297Q, D265A, C127S, S228P, and combinations thereof. In some embodiments, the mutation is N297A, N297Q, D265A, or a combination thereof. In some embodiments, the mutation is C127S. In some embodiments, the mutation is S228P. In certain embodiments, the heavy chain includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In certain embodiments, the heavy chain is selected from the group consisting of immunoglobulins IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In certain embodiments, the immunoglobulin is a human immunoglobulin. In certain embodiments, the heavy chain is an IgG ₁ heavy chain, which includes a mutation selected from the group consisting of N297A, N297Q, D265A, or a combination thereof. In certain embodiments, the heavy chain is an IgG ₂ heavy chain, which includes the C127S mutation. In certain embodiments, the heavy chain is an IgG ₄ heavy chain, which includes the S228P mutation. In certain embodiments, the heavy chain is an IgG ₁ heavy chain, which includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In certain embodiments, the resistance system is antagonistic.

In a specific aspect, an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes: a first antigen-binding domain that binds to OX40, as described herein; and a second antigen-binding domain that does not It will specifically bind to an antigen expressed by human immune cells (ie, the second antigen binding domain will not bind to OX40 or any other antigen expressed by human immune cells), as described herein. In certain embodiments, the first and second antigen binding domains include complementary CH3 domains. For example, complementary CH3 domains allow heterodimerization to occur preferentially between the heavy chain of the first antigen-binding domain and the heavy chain of the second antigen-binding domain, rather than homodimerization of the respective antigen-binding domains. Any technology known to those familiar with the technology can be used (including but not limited to, such as in Ridgway, JBB et al., (1996) Protein Eng 9(7): 617-621 and McDonald’s The knob-into-hole technique described in Merchant M et al.) is used to produce complementary CH3 domains. For example, the knob-into-hole technology replaces the small amino acid in the first CH3 domain with a larger amino acid (ie, "overhang") and replaces the large amine in the second CH3 domain. The base acid is replaced with a smaller amino acid (ie "hole"). The polypeptide including the CH3 domain can then be dimerized based on the interaction of the protrusion and the hole. In certain embodiments, one of the antigen-binding domains includes the first IgG ₁ CH3 domain, which includes a substitution selected from the group consisting of T366Y and T366W, and the other antigen-binding domain includes The second IgG ₁ CH3 domain, which includes a substitution selected from the group consisting of Y407T, T366S, L368A, and Y407V. In some embodiments, the antigen bound by the second antigen binding domain is not naturally expressed by human immune cells. In some embodiments, immune cells are selected from the group consisting of: T cells (CD4+ T cells or CD8+ T cells), B cells, natural killer cells, dendritic cells, macrophages, and eosinophils cell. In certain embodiments, the antigen binding domain that specifically binds to OX40 includes a first VH and a first VL, and the second antigen binding domain includes a second VH and a second VL. In certain embodiments, the antigen binding domain that specifically binds to OX40 includes a first heavy chain and a first light chain, and the second antigen binding domain includes a second heavy chain and a second light chain. In certain embodiments, the antibody is used for administration to a sample or subject, wherein the second antigen-binding domain is non-reactive (ie, the antigen bound by the second antigen-binding domain is not present in the sample or subject) . In certain embodiments, the second antigen-binding domain will not specifically bind to an antigen on OX40-expressing cells (eg, the second antigen-binding domain will not bind to an antigen that is naturally expressed by OX40-expressing cells). In certain embodiments, the antibody functions as a monovalent antibody (ie, an anti-OX40 monovalent antibody) in the sample or subject, wherein the first antigen-binding domain of the antibody binds to OX40, and the second antigen-binding domain is in the sample or subject. The subject is non-reactive (e.g., due to the absence of the antigen bound by the second antigen-binding domain in the sample or subject). In some embodiments, the second antigen binding domain specifically binds to a non-human antigen (ie, an antigen that is expressed in other organisms than in humans). In certain embodiments, the second antigen binding domain specifically binds to viral antigens. In certain embodiments, the viral antigen is derived from a virus that does not infect humans (ie, a non-human virus). In certain embodiments, viral antigens are not present in human immune cells (e.g., human immune cells are not infected by viruses associated with viral antigens). In some embodiments, the viral antigen is an HIV antigen. In certain embodiments, the second antigen binding domain specifically binds chicken albumin or chicken egg lysozyme. In certain embodiments, the second antigen binding domain specifically binds to an antigen that is not expressed by (ie, is not present in) wild-type cells (eg, wild-type human cells). In certain embodiments, the second antigen binding domain specifically binds to a tumor-associated antigen that is not expressed by (ie, is not present in) normal cells (e.g., wild-type cells (e.g., wild-type human cells)). In certain embodiments, tumor-associated antigens are not expressed by (ie, are not present in) human cells. In certain embodiments, the heavy chain constant region of the second antigen binding domain includes a mutation selected from the group consisting of N297A, N297Q, D265A, C127S, S228P, and combinations thereof. In some embodiments, the mutation is N297A, N297Q, D265A, or a combination thereof. In some embodiments, the mutation is C127S. In some embodiments, the mutation is S228P. In certain embodiments, the heavy chain constant region of the second antigen binding domain includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In certain embodiments, the heavy chain constant regions of the first and second antigen binding domains are selected from the group consisting of immunoglobulins IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In certain embodiments, the immunoglobulin is a human immunoglobulin. In certain embodiments, the heavy chain constant regions of the first and second antigen binding domains are isotyped. In certain embodiments, the first antigen binding domain includes a first IgG ₁ heavy chain constant region and the second antigen binding domain includes a second IgG ₁ heavy chain constant region, wherein the first and second heavy chain constant regions include selected from The same mutations in the following group, which consists of the following: N297A, N297Q, D265A, or a combination thereof. In certain embodiments, the first antigen binding domain includes a first IgG ₁ heavy chain constant region and the second antigen binding domain includes a second IgG ₁ heavy chain constant region, wherein the first and second heavy chain constant regions include selected from The same mutations in the following group, which consists of the following: D265A, P329A, or a combination thereof. In certain embodiments, the first antigen binding domain includes a first IgG ₂ heavy chain constant region and the second antigen binding domain includes a second IgG ₂ heavy chain constant region, wherein the first and second heavy chain constant regions include the C127S mutation . In certain embodiments, the first antigen binding domain includes a first IgG ₄ heavy chain constant region and the second antigen binding domain includes a second IgG ₄ heavy chain constant region, wherein the first and second heavy chain constant regions include the S228P mutation . In certain embodiments, the resistance system is antagonistic.

In a specific aspect, an antibody that immunospecifically binds to OX40 (eg, human OX40) as described herein includes: a first antigen-binding domain that specifically binds to OX40, which includes a first heavy chain and a first light chain; and Double chain or fragments thereof. In certain embodiments, the first and second heavy chains, or fragments of the second heavy chain, include complementary CH3 domains. For example, complementary CH3 domains allow heterodimerization to occur preferentially between the heavy chains rather than homodimerization of the respective heavy chains. In certain embodiments, one of the heavy chains includes the first IgG ₁ CH3 domain, which includes a substitution selected from the group consisting of T366Y and T366W, and the other heavy chain includes the second IgG ₁ CH3 domain, which includes substitutions selected from the group consisting of Y407T, T366S, L368A, Y407V. In some embodiments, the fragment of the second heavy chain is an Fc fragment. In certain embodiments, the second heavy chain or a fragment thereof is derived from an antigen binding domain that specifically binds to a non-human antigen (ie, an antigen that is expressed in other organisms other than humans). In certain embodiments, the second heavy chain or a fragment thereof is derived from an antigen binding domain that specifically binds viral antigens. In certain embodiments, viral antigens are not present in human immune cells (e.g., human immune cells are not infected by viruses associated with viral antigens). In some embodiments, the viral antigen is an HIV antigen. In certain embodiments, the second heavy chain or a fragment thereof is derived from an antigen binding domain that specifically binds to chicken albumin or chicken egg lysozyme. In certain embodiments, the second heavy chain or fragment thereof is derived from an antigen binding domain that specifically binds to an antigen that is not expressed by (ie, is not present in) wild-type cells (eg, wild-type human cells). In certain embodiments, the second heavy chain or fragment thereof is derived from an antigen that specifically binds to a tumor-associated antigen that is not expressed by (ie, is not present in) normal cells (e.g., wild-type cells (e.g., wild-type human cells)) Combine domain. In certain embodiments, tumor-associated antigens are not expressed by (ie, are not present in) human cells. In certain embodiments, the second heavy chain or a fragment thereof includes a mutation selected from the group consisting of N297A, N297Q, D265A, C127S, S228P, and combinations thereof. In some embodiments, the mutation is N297A, N297Q, D265A, or a combination thereof. In some embodiments, the mutation is C127S. In some embodiments, the mutation is S228P. In certain embodiments, the second heavy chain or a fragment thereof includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In certain embodiments, the first and second heavy chain constant regions are selected from the group consisting of immunoglobulins IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In certain embodiments, the immunoglobulin is a human immunoglobulin. In certain embodiments, the first and second heavy chain constant regions are isotyped. In certain embodiments, the first and second heavy chain constant regions are IgG ₁ constant regions and include the same mutation selected from the group consisting of: N297A, N297Q, D265A, or a combination thereof . In certain embodiments, the first and second heavy chain constant regions are IgG ₁ constant regions and include the same mutation selected from the group consisting of D265A, P329A, or a combination thereof. In certain embodiments, the first and second heavy chain constant regions are IgG ₂ heavy chain constant regions and include the C127S mutation. In certain embodiments, the first and second heavy chain constant regions are IgG ₄ heavy chain constant regions and include the S228P mutation. In certain embodiments, the resistance system is antagonistic.

The above-mentioned reference includes an antigen-binding domain that specifically binds to OX40 (for example, human OX40) and a second antigen-binding domain or second heavy chain In the aspect of antibodies or fragments thereof, the antigen binding domain may include any or any of the anti-OX40 sequences described herein. In certain embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes: (a) the first heavy chain variable domain (VH) including the VH complementarity determining region (CDR) 1, the VH CDR1 Including the amino acid sequence of GSAMH (SEQ ID NO: 4); VH CDR2, which includes the amino acid sequence of RIRSKANSYATAYAASVKG (SEQ ID NO: 5); and VH-CDR3, which includes the amino acid sequence of GIYDSSGYDY (SEQ ID NO: 6) Amino acid sequence; and (b) the first light chain variable domain (VL) including VL-CDR1, which includes the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO:1); VL-CDR2, which includes LGSNRAS (SEQ ID NO: 2) the amino acid sequence; and VL-CDR3, which includes the amino acid sequence of MQALQTPLT (SEQ ID NO: 3). In certain embodiments, the antigen binding domain that specifically binds to OX40 (e.g., human OX40) specifically binds to the same epitope of OX40 (e.g., human OX40) as an antibody including: VH, which includes SEQ ID NO ：16, and VL, which includes the amino acid sequence of SEQ ID NO:15. In certain embodiments, the antigen binding domain that specifically binds to OX40 (e.g., human OX40) displays the same protein as SEQ ID NO: 55 (except for the presence of From the lower group of amino acid mutations, the group consists of the following: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and combinations thereof) reduced binding or no binding. In certain embodiments, the antigen binding domain that specifically binds to OX40 (e.g., human OX40) includes VH and VL, where VH includes SEQ ID NO: 16 amino acid sequence. In certain embodiments, the antigen binding domain that specifically binds to OX40 (eg, human OX40) includes VH and VL, where VL includes the amino acid sequence of SEQ ID NO:15. In certain embodiments, the antigen binding domain that binds to OX40 includes: VH, which includes at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 16 The amino acid sequence. In some embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes: VH, which includes the amino acid sequence of SEQ ID NO:16. In certain embodiments, the antigen binding domain that binds to OX40 includes: VH, which includes a germline sequence derived from human IGHV3-73 (e.g., IGHV3-73*01 (e.g., having the amino acid sequence of SEQ ID NO: 19) )) of the amino acid sequence. In certain embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes: VL, which includes at least 75%, 80%, 85%, 90% of the amino acid sequence of SEQ ID NO: 15 , 95%, or 99% identical amino acid sequence. In certain embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes: VL-CDR3, which includes the amino acid sequence SEQ ID NO:3. In certain embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes: VL, which includes the amino acid sequence of SEQ ID NO:15. In certain embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes a light chain, which includes the amino acid sequence of SEQ ID NO:20. In some embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes a light chain, which includes the amino acid sequence of SEQ ID NO:50. In certain embodiments, it specifically binds to OX40 (e.g., human The antigen binding domain of OX40) includes: VL, which includes an amino acid sequence derived from the germline sequence of human IGKV2-28 (for example, IGKV2-28*01 (for example, having the amino acid sequence of SEQ ID NO: 18)) . In certain embodiments, the antigen binding domain that specifically binds to OX40 (e.g., human OX40) includes the VH and VL sequences listed in SEQ ID NOs: 16 and 15, respectively. In some embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes a heavy chain, which includes the amino acid sequence of SEQ ID NO:21. In some embodiments, the antigen binding domain that specifically binds to OX40 (for example, human OX40) includes a heavy chain, which includes the amino acid sequence of SEQ ID NO:60. In certain embodiments, the antigen binding domain that specifically binds to OX40 (eg, human OX40) includes a mutation selected from the group consisting of N297A, N297Q, D265A mutations, or a combination thereof. In certain embodiments, the antigen binding domain that specifically binds to OX40 (e.g., human OX40) includes a mutation selected from the group consisting of D265A, P329A, and combinations thereof.

In certain embodiments, the antagonist antibodies described herein antagonize OX40 (e.g., human OX40). In certain embodiments, the antibody inactivates, reduces or inhibits the activity of OX40 (e.g., human OX40). In certain embodiments, the antibody inhibits or reduces the binding of OX40 (e.g., human OX40) to OX40 ligand (e.g., human OX40 ligand). In certain embodiments, the antibody inhibits or reduces OX40 (e.g., human OX40) signaling. In certain embodiments, the antibody inhibits or reduces the induction of OX40 ligand (e.g., human OX40 ligand) Guided OX40 (e.g., human OX40) activity (e.g., OX40 signaling). In certain embodiments, the antagonist antibodies described herein inhibit or reduce T cell proliferation. In certain embodiments, the antagonist antibodies described herein inhibit or reduce T cell proliferation. In certain embodiments, the antagonist antibodies described herein inhibit or reduce the production of cytokines (e.g., inhibit or reduce IL-2, TNFα, IFNγ, IL-4, IL-10, IL-13, or a combination thereof). In certain embodiments, the antagonist antibodies described herein inhibit or reduce IL-2 production by SEA-stimulated T cells. In certain embodiments, the antagonist antibodies described herein block the interaction of OX40 and OX40L (eg, block the binding of OX40L and OX40 to each other (eg, block the binding of human OX40 ligand to human OX40)) .

In certain embodiments, relative to the activity of OX40 (e.g., human OX40) in the absence or presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), immunospecific binding to OX40 (E.g., human OX40) antagonist antibodies described herein reduce the activity of OX40 (e.g., human OX40) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold , 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 Times or 100 times, as determined by methods described herein and/or known to those skilled in the art. In certain embodiments, compared to OX40 (e.g., human OX40) activity, the antagonist antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) reduces the activity of OX40 (e.g., human OX40) by at least about 5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, such as Measured by methods described herein and/or known to those skilled in the art. Non-limiting examples of OX40 (e.g., human OX40) activity can include OX40 (e.g., human OX40) signaling, cell proliferation, cell survival, and cytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13). In certain embodiments, the antagonist antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) inhibit, reduce, or inactivate OX40 (e.g., human OX40) activity. In a specific embodiment, OX40 activity is determined as described in the examples below.

In certain aspects, the antagonist antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) inhibit, reduce, or inactivate the cell proliferation of cells that express OX40 and respond to OX40 signaling (e.g., in response to OX40 Cells that proliferate by stimulating and OX40 signaling (such as T cells). The cell proliferation assay is as described in the art (for example, ³ H-thymidine incorporation method, BrdU incorporation method, or CFSE method, as described in the following examples), and can be easily performed by those skilled in the art Implement. In a specific embodiment, compared to using only T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex stimulating agent (Such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells, using T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) , Or TCR complex stimulating antibody (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) in the immune specific binding OX40 (such as human OX40) described here In the presence of antagonistic antibodies, cell proliferation is reduced.

In certain aspects, the antagonist antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) reduce the survival of cells (e.g., T cells (e.g., CD4 and CD8 effector T cells)). In a specific embodiment, compared to T cells stimulated with only T cell mitogens, T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol) are used. Ethyl ester (PMA), or TCR complex stimulating antibody (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (for example, CD4 ⁺ or CD8 ⁺ effector T cells) in the immune specific binding OX40 (for example, human OX40 ) Has reduced survival in the presence of the antagonist antibodies described herein. The cell survival assay is as described in the art (for example, trypan blue exclusion assay) and can be easily performed by a person familiar with the technique.

In a specific embodiment, an antagonist antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) reduces cell survival (e.g., T cells (such as CD4 and CD8 effector T cells)) by at least about 1.2-fold, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times , 30 times, 40 times, 50 times, 60 times, 70-fold, 80-fold, 90-fold, or 100-fold, as described herein or known to those skilled in the art (for example, trypan blue exclusion assay), in the absence of any antibody or the presence of irrelevant antibodies ( For example, it is determined in the case of an antibody that does not immunospecifically bind to OX40). In a specific embodiment, relative to the activity of OX40 (e.g., human OX40) in the absence of any antibody or the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to OX40), immunospecific binding to OX40 ( For example, the human OX40) antagonist antibodies described herein reduce cell survival (e.g., T cells (such as CD4 and CD8 effector T cells)) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, such as It is measured by a method described herein or known to those skilled in the art (for example, trypan blue exclusion assay).

In some embodiments, compared to stimulating the antibody with only T cell mitogens or T cell receptor complex stimulators, such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex (Such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells, T cells stimulated with T cell mitogens (for example, anti-CD3 antibodies or phorbol esters) (for example, CD4 ⁺ or CD8 ⁺ effector T cells) In the presence of the antagonist antibodies described herein that immunospecifically bind to OX40 (eg, human OX40), there is a reduction of at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold Times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90-fold or 100-fold cell survival, as determined by methods described herein or known to those skilled in the art (for example, trypan blue exclusion assay). In some embodiments, compared to T cells stimulated with only T cell mitogens, T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol beta) are used. Ester (PMA), or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) to specifically bind to OX40 (such as human OX40) In the presence of the antagonist antibodies described herein, there is a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of cell survival, as described herein or by methods known to those skilled in the art ( For example, trypan blue exclusion assay).

In certain embodiments, the antagonist antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) cannot prevent effector T cells (e.g., CD4 ⁺ and CD8 ⁺ effector T cells) from priming-induced cell death.

In a specific embodiment, compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), in the presence or absence of OX40 (e.g., human OX40) stimulation Cytokines are produced, and the antagonistic antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) produce cytokines (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL- 10, and/or IL-13) inhibited, reduced, or inactivated at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% Or 99%, as determined by methods described here (see the examples below) or by methods known to those skilled in the art. In a specific embodiment, compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to OX40), in the presence or absence of OX40 (e.g., human OX40) stimulation Cytokines are produced, and the antagonistic antibodies described herein that immunospecifically bind to OX40 (e.g., human OX40) produce cytokines (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL- 10. and/or IL-13) inhibit or reduce at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times , 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as described by ( See the following examples, as measured in Example 2) or methods known to those skilled in the art.

In certain embodiments, compared to only stimulating T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex stimulation T cells stimulated by antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies), use T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) ), or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti-CD28 antibodies)) stimulated T cells (for example, CD4 ⁺ or CD8 ⁺ effector T cells) that immunospecifically bind to OX40 (for example, human OX40). In the presence of the described antagonistic antibody, it has a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of cytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13), as determined by methods described herein or known to those skilled in the art (e.g., ELISA assay or as described in the examples below). In some embodiments, compared to stimulating the antibody with only T cell mitogens or T cell receptor complex stimulators, such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA), or TCR complex (Such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells, using T cell mitogens or T cell receptor complex stimulators (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA) , Or TCR complex stimulating antibody (such as anti-CD3 antibody and anti-CD28 antibody)) stimulated T cells (such as CD4 ⁺ or CD8 ⁺ effector T cells) in the immune specific binding OX40 (such as human OX40) described here In the presence of the antagonistic antibody, it has a reduction of at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times Times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times the production of cytokines (e.g., IL- 2. TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13), as described herein or known to those skilled in the art (for example, ELISA assay or as follows As described in the examples) determined.

In a specific embodiment, compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., does not immunospecifically bind to OX40 The antagonist antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) reduces IL-2 production in response to staphylococcal enterotoxin A (SEA) stimulation. At least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, as described here (see the following examples, such as example 2) or familiar with the technology Measured by a known method.

In certain embodiments, T cells stimulated with staphylococcal enterotoxin A (SEA) (eg, CD4 ⁺ or CD8 ⁺ T cells) specifically bind to OX40 ( For example, human OX40) in the presence of the antagonistic antibody described herein has a reduction of at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold Times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times IL-2 production, as measured by methods described herein or known to those skilled in the art (e.g., ELISA assay or as described in the examples below).

The anti-OX40 antibody can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels (e.g., glucose oxidase); radioisotopes (e.g., iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and Tu ( ⁹⁹ Tc)); luminescent labels (such as luminol); and fluorescent labels (such as luciferin and rhodamine and biotin). Such labeled antibodies can be used to detect OX40 (e.g., human OX40) protein. See, for example, section 5.5.2 below.

5.3 Antibody production

Antibodies that immunospecifically bind to OX40 (e.g., human OX40) can be produced by any method known in the art to synthesize antibodies, for example, by chemical synthesis or by recombinant expression technology. The methods described here adopt, unless otherwise specified, molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization and related fields in the technical field Conventional technology. These techniques are described in, for example, the references cited herein and are fully explained in the literature. See, for example, Maniatis T et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sam Sambrook J et al., (1989), Molecular Clones: Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001), Molecular Clones: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Handbook of Contemporary Molecular Biology Experiments, John & Wiley & Sons (1987 and updated annually); Handbook of Contemporary Immunology Experiments (Current Protocols in Immunology) , John & Wiley & Sons (1987 and updated annually) Gait (Edit) (1984) Oligonucleotide Synthesis: A Practical Approach (Oligonucleotide Synthesis: A Practical Approach), IRL Press; Aix Eckstein (Edit) (1991) Oligonucleotides and Analogues: A Practical Approach (Oligonucleotides and Analogues: A Practical Approach), IRL Press; Birren B et al., (Edit) (1999) Genome Analysis: Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In a specific embodiment, the antibody system described herein is an antibody (e.g., recombinant antibody) prepared, expressed, created, or isolated by any means involving creation (e.g., through DNA sequence synthesis, genetic engineering). In certain embodiments, such antibodies include sequences (e.g., DNA sequences or amino acid sequences) that are not naturally found in the germline of antibodies in animals or mammals (e.g., humans).

In a certain aspect, provided herein is a method of making an antibody that immunospecifically binds to OX40 (e.g., human OX40), the method comprising culturing the cells or host cells described herein. In a certain aspect, provided herein is a method of making antibodies that immunospecifically bind to OX40 (e.g., human OX40), the method comprising using the cells or host cells described herein (e.g., cells or host cells, which include encoding The antibody (polynucleotide) described herein expresses (e.g., recombinant expresses) the antibody. In a specific embodiment, the cell line is an isolated cell. In a specific embodiment, the exogenous polynucleotide has been introduced into the cell. In a specific embodiment, the method further includes the step of purifying the antibody obtained from the cell or host cell.

Methods for producing multiple strains of antibodies are known in the art (see, for example, the Short Protocols in Molecular Biology), (2002) fifth edition, Ausubel FM et al., editor, John & Wiley & Sons, New York, Chapter 11).

Various techniques known in the art can be used to prepare monoclonal antibodies, including the use of fusion tumors, recombinants, and phage display technologies or combinations thereof. For example, fusionoma technology can be used to produce monoclonal antibodies, including techniques known and taught in the art, for example, in Harlow E and Lane D, Antibodies: Laboratory Manual (Antibodies: A Laboratory Manual), Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling (Hammerling) GJ et al. in Monoclonal Antibodies and T-Cell Hybridomas (Monoclonal Antibodies and T-Cell Hybridomas) 563 681 (Love Elsevier (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by fusionoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells that exogenously express the antibodies described herein.

In a specific embodiment, a "monoclonal antibody" as used herein is an antibody produced by a single cell (for example, a fusionoma or host cell that produces a recombinant antibody), wherein, for example, by ELISA or known in the art or Other antigen-binding or competitive binding analyses in the embodiments provided herein are used to determine that the antibody immunospecifically binds to OX40 (e.g., human OX40). In a specific embodiment, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody system is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In some embodiments, the monoclonal antibody may be a Fab fragment or a F(ab') ₂ fragment. It can be manufactured, for example, by the fusion tumor method as described in Kohler G and Milstein C (1975) Nature 256:495 or can be, for example, obtained from a phage library using a technique as described herein. Isolate the monoclonal antibodies described here. Other methods for preparing clone cell lines and monoclonal antibodies expressed by them are well-known in the art (see, for example, the above-mentioned refined molecular biology experiment guide, (2002) 5th edition, Ausubel Chapter 11 in FM et al.).

Methods of using fusionoma technology for the production and screening of specific antibodies are routine and well known in the art. For example, in the fusion tumor method, mice or other appropriate host animals (such as sheep, goats, rabbits, rats, hamsters, or rhesus monkeys) are immunized to initiate or produce proteins that specifically bind for immunization (such as , OX40 (e.g., human OX40)) antibodies to lymphocytes. Alternatively, lymphocytes can be immunized in vitro. Then, use a suitable flux (such as polyethylene glycol) to fuse lymphocytes with myeloma cells to form fusion tumor cells (Goding JW (Edit), Monoclonal Antibodies: Principles and Practices (Monoclonal Antibodies: Principles and Practice) Practice), pages 59-103 (Academic Press, 1986)). In addition, RIMMS (Multiple Site Repeat Immunization) technology can be used to immunize animals (Kilpatrick KE et al. (1997) Hybridoma 16:381-9, which is incorporated by reference in its entirety) .

In some embodiments, mice (or other animals, such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., OX40 (e.g., human OX40)), and once detected For the immune response, for example, antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is collected and the spleen cells are isolated. Then, the spleen cells are fused to any appropriate myeloma cells by well-known techniques, such as those from the cell line SP20 (available from the American Germplasm Conservation Center (ATCC ^® ) (Manassas, VA)) Cells to form fusion tumors. Selection and selection of fusion tumors by limited dilution. In certain embodiments, lymph nodes of immunized mice are collected and fused with NS0 myeloma cells.

Therefore, the prepared fusion tumor cells are seeded and grown in an appropriate medium, which preferably contains one or more substances capable of inhibiting the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the fusion tumor will typically include hypoxanthine, aminopterin, and thymidine (HAT medium). The substance prevents the growth of HGPRT-deficient cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Use of myeloma cells that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium (such as HAT medium). Among these, myeloma cell lines are murine myeloma lines, such as NS0 cell lines or cell lines derived from MOPC-21 and MPC-11 mouse tumors, which can be obtained from Holy Land in California (CA), USA Salk Institute Cell Distribution Center (San Diego), and SP-2 or X63-Ag8.653 cells, which can be obtained from Rockville, Maryland (MD) ) American species Quality preservation center. The use of human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies is also described (Kozbor D (1984) Journal of Immunology 133: 3001-5; Brodie (Brodeur) ) Et al., Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987).

In order to generate monoclonal antibodies against OX40 (e.g., human OX40), the culture medium in which the fusion tumor cells are grown is analyzed. Determine the binding specificity of monoclonal antibodies produced by fusion tumor cells by methods known in the art, for example, immunoprecipitation or in vitro binding identification methods, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay Method (ELISA).

After identifying fusion tumor cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be sub-colonized by a limiting dilution process and standard methods (Godin JW above (Editor) , Monoclonal antibody: principle and practice) to make it grow. For this purpose, suitable media include, for example, D-MEM or RPMI 1640 media. In addition, fusion tumor cells can grow in vivo as ascites tumors in animals.

By conventional immunoglobulin purification procedures (for example, protein A-cross-linked agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography), the monoclonal antibodies secreted by the subgenes are appropriately removed from Separate from culture medium, ascites fluid or serum.

The antibodies described herein can be produced by any technique known to those skilled in the art. For example, enzymes (such as papain (to produce Fab fragments)) or pepsin (to produce F(ab') ₂ fragments) can be produced by proteolytic cleavage of immunoglobulin molecules to produce the Fab and F(ab ') ₂ fragments. The Fab fragment corresponds to one of the two identical arms of the tetrameric antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab') ₂ fragment contains two antigen-binding arms of a tetrameric antibody molecule connected by disulfide bonds in the hinge region.

In addition, various phage display methods known in the art can also be used to generate the antibodies described herein. In phage display methods, proteins are displayed on the surface of phage particles, which carry polynucleotide sequences encoding them. Specifically, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or murine affected tissue cDNA library). The DNA encoding the VH and VL domains were recombined with the scFv linker by PCR, and cloned into the phagemid vector. The vector was transferred to E. coli by electroporation, and the E. coli was infected with helper phage. The phage used in these methods is typically a filamentous phage including fd and M13, and the VH and VL domains are usually recombinantly fused with phage gene III or gene VIII. Phages that express antibodies that bind to a specific antigen can be selected or identified using antigens (for example, using labeled antigens or antigens that are bound or captured to a solid surface or beads). Examples of phage display methods that can be used to prepare the antibodies described herein include those disclosed in: Brinkman et al. (1995) J Immunol Methods 182: 41-50; Aim (Ames) RS et al. (1995) Journal of Immunological Methods 184:177-186; Kettleborough CA et al. (1994) Eur J Immunol 24:952-958; Persic L et al. (1997) Gene 187: 9-18; Burton DR and Barbas CF (1994), Advan Immunol 57: 191 -280; PCT application number PCT/GB 91/001134; international publication number WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95 /20401, and WO 97/13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,108, and 5,969,743.

As explained in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce antibodies (including human antibodies), and in any desired host (including breastfeeding It is expressed in animal cells, insect cells, plant cells, yeast, and bacteria (e.g., as described below). Techniques for recombinant production of antibodies (such as Fab, Fab' and F(ab') ₂ fragments) can also be used, using methods known in the art, such as those disclosed in the following: PCT Publication No. WO 92/ 22324; Mullinax RL et al. (1992) BioTechniques 12(6): 864-9; Sawai H et al. (1995) American Journal of Reproductive Immunology (Am J Reprod Immunol 34: 26-34; and Better M et al. (1988) Science 240: 1041-1043.

In one aspect, in order to generate antibodies, PCR primers including VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify VH or VL sequences from templates (eg, scFv clones) . Using the selection techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing VL Constant region (for example, human kappa or lambda constant region) in a vector. The VH and VL domains can also be cloned into a vector that exhibits the necessary constant regions. Then, using techniques known to those skilled in the art, the heavy chain transformation vector and the light chain transformation vector are co-transfected into the cell line to produce a stable or transient cell line expressing antibodies (eg, IgG).

Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT and Morrison SL (1986) Biotechnology 4: 214-221; Gillies SD et al. , (1989) Journal of Immunological Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

The humanized antibody can bind to a predetermined antigen and includes a framework region, which has substantially the amino acid sequence of a human immunoglobulin, while the CDR has substantially the amino acid sequence of a non-human immunoglobulin (for example, murine immunoglobulin) Acid sequence. In a specific embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), which is usually a human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ . Various techniques known in the art can be used to produce humanized antibodies, including, but not limited to, CDR-grafting (European Patent No. EP 239400; International Publication No. WO 91/09967; and U.S. Patent No. 5,225,539, 5,530,101, And 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4/5): 489 -498; Studnicka GM et al. (1994) Prot Engineering 7(6): 805-814; and Roguska MA et al. (1994) Proceedings of the National Academy of Sciences 91 : 969-973), chain shuffling (U.S. Patent No. 5,565,332), and the technology disclosed in the following items: for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 93/ 17105; Tan (Tan) P et al. (2002) Journal of Immunology 169:1119-25; Caldas C et al. (2000) Protein Engineering 13(5):353-60; Moreia ( Morea) V et al. (2000) Methods 20(3): 267-79; Baca M et al. (1997) J Biol Chem 272(16): 10678-84 ; Roguska MA et al. (1996) Protein Engineering 9(10): 895 904; Couto JR et al. (1995) Cancer Research 55 (Supplement 23): 5973s-5977s; Coto (Couto) JR et al. (1995) Cancer Research 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al. , (1994) J Mol Biol 235(3):959-73. See also U.S. Application Publication No. US 2005/0042664 A1 (February 24, 2005), which application is incorporated herein by reference in its entirety.

Single domain antibodies (for example, antibodies lacking light chains) can be produced by methods well known in the art. See Riechmann L and Muyldermans S (1999) Journal of Immunology 231: 25-38; Nuttall SD et al. (2000) Curr Pharm Biotechnol ) 1(3): 253-263; Maudmans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 6,005,079; and International Publication No. WO 94/04678 , WO 94/25591 and WO 01/44301.

In addition, antibodies that immunospecifically bind to the OX40 antigen can then be used to generate anti-idiotypic antibodies that "mock" the antigen, using techniques well known to those skilled in the art. (See, for example, Greenspan (Greenspan) NS and Bona (Bona) CA (1989) FASEB Journal 7(5):437-444; and Nissinoff A (1991) Journal of Immunology 147(8 ): 2429-2438).

In a specific embodiment, the anti-system human antibody described herein that binds to the same epitope of OX40 (e.g., human OX40) as the anti-OX40 antibody described herein. In a specific embodiment, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) any of the antibodies described herein (e.g., pab1949 or pab2044) binds to OX40 (e.g., human OX40) System human antibodies. Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulin but can express human immunoglobulin genes can be used. Specifically, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and variable regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be introduced into the human immunoglobulin locus separately or by homologous recombination and become non-functional at the same time. Specifically, _{the homozygous deletion of the JH} region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to produce chimeric mice. Then, the chimeric mice are raised to produce homozygous offspring that express human antibodies. The selected antigen is used to immunize the transgenic mouse in a normal manner (e.g., all or part of the antigen (e.g., OX40)). Conventional fusion tumor technology can be used to obtain monoclonal antibodies against antigens from immunized transgenic mice. The human immunoglobulin transgene carried by the transgenic mice rearranges during B cell differentiation, and then undergoes class switching and somatic mutation. Therefore, using this technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for the production of human antibodies, see Lonberg N and Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and the procedures for producing such antibodies, see, for example, International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735; And U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capable of producing human antibodies include Xenomouse ^TM (Abgenix (Abgenix); U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse ^TM (Mederex, Inc.)/Gen Pharm; US Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse ^TM (Kirin), and KM Mouse ^TM (Medarex/Kirin).

Human antibodies that specifically bind to OX40 (eg, human OX40) can be produced by a variety of methods known in the art, including the phage display method described above, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

In some embodiments, mouse-human fusion tumors can be used to generate human antibodies. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human fusion tumors that secrete human monoclonal antibodies, and these mouse-human fusion tumors can be screened In order to determine the fusionoma secreting human monoclonal antibodies that immunospecifically bind to the target antigen (e.g., OX40 (e.g., human OX40)). Such methods are known and described in the art, see, for example, Shinmoto H et al. (2004) Cytotechnology 46:19-23; Naganawa Y Et al. (2005) Human Antibodies 14:27-31.

5.3.1 Polynucleotides

In certain aspects, polynucleotides are provided herein, which include the antibodies or fragments thereof (e.g., variable light chain region and/or can The nucleotide sequence of the variable heavy chain region), and vectors, such as vectors including such polynucleotides, are used for recombinant expression in host cells (e.g., Escherichia coli and mammalian cells). A polynucleotide is provided herein, which includes a nucleotide sequence encoding any of the antibodies provided herein, together with a vector, which includes such a polynucleotide sequence, for example, is effectively expressed in a host cell (e.g., a mammalian cell) Their performance vehicles.

As used herein, an "isolated" polynucleotide or nucleic acid molecule is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule (e.g., in mice or humans). In addition, "isolated" nucleic acid molecules (such as cDNA molecules) may be substantially free of other cellular materials or when produced by recombinant technology, without culture media, or when chemically synthesized, are substantially free of chemical precursors or other chemical agents. . For example, the language "substantially free" includes other materials having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) (e.g., Materials, culture media, other nucleic acid molecules, chemical precursors and/or other chemical agents) Preparation of polynucleotides or nucleic acid molecules. In a specific embodiment, one or more nucleic acid molecules encoding the antibodies described herein are separated or purified.

In a specific aspect, polynucleotides are provided herein, which include nucleotide sequences encoding antibodies that immunospecifically bind to OX40 polypeptides (e.g., human OX40) and include amino acid sequences as described herein, together with antibodies, It competes with such antibodies that bind to the OX40 polypeptide (e.g., in a dose-dependent manner), or binds to the same epitope as such antibodies.

In certain aspects, polynucleotides are provided herein that include nucleotide sequences encoding the light or heavy chains of the antibodies described herein. A polynucleotide may include a nucleotide sequence encoding a light chain (including the VL FR and CDR of the antibodies described herein (see, for example, Tables 1 and 3)). The polynucleotide may include a nucleotide sequence encoding a heavy chain (including the VH FR and CDR of the antibodies described herein (see, for example, Tables 2 and 4)). In a specific embodiment, the polynucleotide described herein encodes a VL domain, which includes the amino acid sequence set forth in SEQ ID NO:15. In a specific embodiment, the polynucleotide described herein encodes a VH domain, which includes the amino acid sequence set forth in SEQ ID NO:16.

In a specific embodiment, polynucleotides are provided herein, which include encoding including three VL chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3 including any of the antibodies described herein) (e.g., see Table 1) ) The nucleotide sequence of the anti-OX40 antibody. In a specific embodiment, polynucleotides are provided herein that include three VH chain CDRs (e.g., including VH CDR1, VH CDR2, and VH CDR3 of any of the antibodies described herein (e.g., see Table 2)). On the specific implementation side In the formula, a polynucleotide is provided here, which includes a nucleotide sequence encoding an anti-OX40 antibody that includes three VH chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR2 of any of the antibodies described herein). CDR3 (for example, see Table 1)) and three VH chain CDRs (for example, including the VH CDR1, VH CDR2, and VH CDR3 of any of the antibodies described herein (for example, see Table 2)).

In a specific embodiment, a polynucleotide is provided herein, which includes a nucleotide sequence encoding an anti-OX40 antibody or fragment thereof, the antibody or fragment thereof includes a VL domain, such as FR1-CDR1-FR2-CDR2-FR3 -CDR3-FR4, including the amino acid sequences described herein (see, for example, Tables 1 and 3, for example, the VL CDR and VLFR of the specific antibody identified in the table by name). In a specific embodiment, a polynucleotide is provided herein, which includes a nucleotide sequence encoding an anti-OX40 antibody or fragment thereof, the antibody or fragment thereof includes a VH domain, such as FR1-CDR1-FR2-CDR2-FR3 -CDR3-FR4, including the amino acid sequences described herein (see, for example, Tables 2 and 4, for example, the VH CDR and VH FR of the specific antibody identified in the table by name).

In certain embodiments, the polynucleotide described herein includes a nucleotide sequence encoding the antibody provided herein, the antibody includes a light chain variable region that includes the amino acid sequence described herein (e.g., SEQ ID NO: 15), wherein the antibody immunospecifically binds to OX40 (e.g., human OX40). In one embodiment, the polynucleotide described herein includes a nucleotide sequence encoding the antibody pab1949 or pab2044 or a fragment thereof provided herein, the antibody or fragment thereof includes a light chain variable region, and the light chain is variable The region includes the amino acid sequence described herein (e.g., SEQ ID NO: 15).

In certain embodiments, the polynucleotide described herein includes a nucleotide sequence encoding the antibody provided herein, the antibody including a heavy chain variable region including the amino acid described herein Sequence (e.g., SEQ ID NO: 16), wherein the antibody immunospecifically binds to OX40 (e.g., human OX40). In one embodiment, the polynucleotide described herein includes a nucleotide sequence encoding the antibody pab1949 or pab2044 or a fragment thereof provided herein, the antibody or fragment thereof includes a heavy chain variable region, and the heavy chain is variable The region includes the amino acid sequence described herein (e.g., SEQ ID NO: 16).

In certain aspects, the polynucleotide includes a nucleotide sequence encoding the antibody or fragment thereof described herein, the antibody or fragment thereof includes a VL domain, and the VL domain includes one or more amines having the amines described herein. The VL FR of the base acid sequence (e.g., see Table 3), wherein the antibody immunospecifically binds to OX40 (e.g., human OX40). In certain aspects, the polynucleotide includes a nucleotide sequence encoding the antibody or fragment thereof described herein, the antibody or fragment thereof includes a VH domain, the VH domain includes one or more amines having the amines described herein. The VH FR of the base acid sequence (e.g., see Table 4), wherein the antibody immunospecifically binds to OX40 (e.g., human OX40).

In a specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding the antibody or fragment thereof described herein, the antibody or fragment thereof includes: a framework region that is a human framework region (for example, a VL domain) And the framework region of the VH domain), wherein the antibody immunospecifically binds to OX40 (e.g., human OX40). In certain embodiments, the polynucleotides provided herein include nucleotide sequences encoding the antibodies or fragments thereof (e.g., CDRs or variable domains) described in Section 5.2 above.

In specific aspects, polynucleotides are provided herein, which include nucleotide sequences encoding antibodies that include light and heavy chains (e.g., light and heavy chains, respectively). Regarding the light chain, in a specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding a kappa light chain. In another specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding a lambda light chain. In yet another specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding an antibody described herein including a human kappa light chain or a human lambda light chain. In a specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding an antibody that immunospecifically binds to OX40 (e.g., human OX40), wherein the antibody includes a light chain, and wherein the amino group of the VL domain The acid sequence may include the amino acid sequence listed in SEQ ID NO: 15, and wherein the constant region of the light chain includes the amino acid sequence of the human kappa light chain constant region. In another specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding an antibody that immunospecifically binds to OX40 (e.g., human OX40) and includes a light chain, wherein the amino acid of the VL domain The sequence may include the amino acid sequence listed in SEQ ID NO: 15, and wherein the constant region of the light chain includes the amino acid sequence of the human lambda light chain constant region. For example, human constant region sequences may be those described in U.S. Patent No. 5,693,780.

In a specific embodiment, the polynucleosides provided herein The acid includes a nucleotide sequence encoding the antibody described herein that immunospecifically binds to OX40 (e.g., human OX40), where the antibody includes a heavy chain, and the amino acid sequence of the VH domain may be included in SEQ ID NO: The amino acid sequence listed in 16, and the constant region of the heavy chain includes the amino acid sequence of the human gamma heavy chain constant region.

In a certain embodiment, the polynucleotide provided herein includes an antibody (e.g., pa91949 or pab2044) that immunospecifically binds to OX40 (e.g., human OX40) as described herein, such as for the VH domain, SEQ ID NO : 16, or for the VL domain, one or more nucleotide sequences of the VH domain and/or VL domain of SEQ ID NO: 15).

In yet another specific embodiment, the polynucleotide provided herein includes a nucleotide sequence encoding the antibody described herein that immunospecifically binds to OX40 (e.g., human OX40), wherein the antibody includes any of the The VL domain and VH domain of the described amino acid sequence, and where the constant region includes the amino acid sequence of human IgG ₁ (for example, allotype 1, 17 or 3), human IgG ₂ or human IgG ₄ constant region.

In a specific embodiment, polynucleotides are provided herein, which include nucleotide sequences encoding anti-OX40 antibodies or domains thereof (as specified herein, see, for example, Tables 1-4, for example antibodies pab1949 or pab2044) .

Also provided herein are polynucleotides encoding anti-OX40 antibodies or fragments thereof optimized by codon/RNA optimization, substitution with heterologous signal sequences, and elimination of mRNA instability elements. By introducing a password The method of modifying and/or eliminating the inhibitory region in mRNA to produce a nucleic acid encoding anti-OX40 antibody or its fragments (for example, light chain, heavy chain, VH domain, or VL domain) with recombination performance optimization can therefore be achieved by The optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498 are performed. For example, potential splice sites and unstable elements in RNA (for example, A/T or A/U-rich elements) can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of RNA recombination performance sex. These changes take advantage of the degeneracy of the genetic code, such as the use of alternative codons for the same amino acid. In some embodiments, it may be desirable to change one or more codons to encode conservative mutations, such as similar amino acids with similar chemical structures and properties and/or functions to the original amino acids.

In certain embodiments, the optimized polynucleotide sequence encoding the anti-OX40 antibody or fragment thereof (e.g., VL domain or VH domain) described herein can be combined with the anti-OX40 antibody or fragment thereof (e.g., , VL domain or VH domain) of the unoptimized polynucleotide sequence of the antisense (e.g., complementary) polynucleotide hybridization. In a specific embodiment, the optimized nucleotide sequence encoding the anti-OX40 antibody or fragment described herein is antisense to the unoptimized polynucleotide sequence encoding the anti-OX40 antibody or fragment thereof under high stringency conditions. Hybridization of polynucleotides. In a specific embodiment, the optimized nucleotide sequence encoding the anti-OX40 antibody or fragment thereof described herein is combined with the anti-OX40 antibody or its fragment encoding the anti-OX40 antibody described herein under high stringency, medium, or lower stringency hybridization conditions. Antisense polynucleotide hybridization of fragments of unoptimized nucleotide sequence. Information about hybridization conditions has been It is described in, for example, U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

The polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. Methods well known in the art can be used to determine the nucleotide sequences that encode the antibodies described herein (for example, the antibodies described in Tables 1-4) and modified forms of these antibodies, that is, to assemble known specific amines in this way. The nucleotide codons of the base acid are used to generate the nucleic acid encoding the antibody. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotide combinations (for example, as described in Kutmeier G et al., (1994), BioTechniques 17:242-246 In), in short, it involves the synthesis of overlapping oligonucleotides comprising encoding antibodies, annealing and ligating those oligonucleotides, and then amplifying the sequence portion of the ligated oligonucleotides by PCR.

Alternatively, methods well known in the art (e.g., PCR and other molecular selection methods) can be used to generate polynucleotides encoding the antibodies or fragments thereof described herein from nucleic acids from suitable sources (e.g., fusionomas). For example, PCR amplification using synthetic primers that can hybridize to the 3'and 5'ends of a known sequence can be performed using genomic DNA obtained from fusion tumor cells that produce the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids that include sequences encoding antibody light and/or heavy chains. Such PCR amplification methods can be used to obtain nucleic acids that include sequences encoding antibody variable light chain regions and/or variable heavy chain regions. The amplified nucleic acid can be cloned into the vector In order to be expressed in host cells and further colonized, for example, to produce chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a specific antibody or fragment thereof is not available, but the sequence of the antibody molecule or fragment thereof is known, the nucleic acid encoding the immunoglobulin or fragment thereof can be chemically synthesized or obtained from a suitable source (for example, antibody cDNA The library is either generated from the following cDNA library, or isolated from the following nucleic acid (preferably poly A+ RNA): any tissue or cell expressing antibodies, such as fusion tumor cells selected to express the antibodies described herein), Amplify by PCR, use synthetic primers that can hybridize to the 3'and 5'ends of the sequence, or by selection, use oligonucleotide probes specific to a specific gene sequence to identify, for example, from a cDNA library CDNA clones encoding antibodies. Then, any method well-known in the art can be used to clone the amplified nucleic acid generated by PCR into a replicable clone vector.

The DNA encoding the anti-OX40 antibody described herein can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the anti-OX40 antibody). Fusion tumor cells can serve as a source of such DNA. Once isolation occurs, the DNA can be placed in the expression vector, and then the vector is transfected into host cells that do not otherwise produce immunoglobulin proteins (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (such as from CHO) GS System ^TM (Lonza's CHO cells) or myeloma cells) to obtain synthetic anti-OX40 antibodies in recombinant host cells.

In order to produce antibodies, including VH or VL nucleotide sequences, The restriction site and PCR primers that protect the flanking sequence of the restriction site can be used to amplify the VH or VL sequence in the scFv clone. Using the selection techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector that expresses the heavy chain constant region (for example, the human γ4 constant region), and the PCR-amplified VH domain can be cloned The VL domain is selected and colonized into a vector that expresses a light chain constant region (for example, a human kappa or lambda constant region). In some embodiments, the vector used to express the VH or VL domain includes an EF-1α promoter, a secretion signal, a selection site for a variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a vector that exhibits the necessary constant regions. Then, using techniques known to those skilled in the art, the heavy chain transformation vector and the light chain transformation vector are co-transfected into the cell line to produce a stable or transient cell line expressing full-length antibodies (eg, IgG).

It is also possible, for example, to modify DNA by substituting murine sequences for the coding sequences of human heavy and light chain constant domains, or by covalently binding all or part of the coding sequences of non-immunoglobulin polypeptides to immunoglobulin coding sequences.

Also provided are polynucleotides that hybridize to polynucleotides encoding the antibodies described herein under high stringency, medium, or lower stringency hybridization conditions. In a specific embodiment, the polynucleotides described herein hybridize to the VH domains provided herein (e.g., SEQ ID NO: 16) and/or under high stringency, medium, or lower stringency hybridization conditions. A polynucleotide of a VL domain (e.g., SEQ ID NO: 15).

Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve the following: Hybridization with DNA bound to the filter membrane in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC/0.1% SDS at about 50°C-65°C ; Hybridization under high stringency conditions may involve the following: hybridization with the nucleic acid bound to the filter in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC/0.2% SDS at about 68°C. Hybrids under other stringent hybridization conditions are known to those familiar with the technique and have been described in, see, for example, Ausubel (Ausubel) FM et al. Edited, (1989) Contemporary Molecular Biology Experiment Manual (Current Protocols in Molecular Biology), Volume I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, Sections 6.3.1-6.3. 6 and 2.10.3 pages.

5.3.2 Cells and vectors

In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) the antibodies described herein that specifically bind to OX40 (e.g., human OX40), as well as related polynucleotides and expression vectors. A vector (for example, an expression vector) is provided herein, the vector includes a polynucleotide including a nucleotide sequence encoding an anti-OX40 antibody or fragment, in a host cell, preferably in a mammalian cell Restructuring performance. Also provided herein are host cells that include such vectors to recombinantly express the anti-OX40 antibodies described herein (e.g., human or humanized antibodies). In a specific aspect, the The antibody method described above, which method comprises expressing such an antibody in a host cell.

The recombinant expression of an antibody or fragment thereof described herein (for example, an antibody heavy chain or light chain or a single chain antibody described herein) that specifically binds to OX40 (for example, human OX40) involves the construction of a performance vector that includes the antibody encoding Or fragments of polynucleotides. Once the polynucleotides encoding the antibodies described herein or fragments thereof (eg, heavy or light chain variable domains) have been obtained, a vector for the production of antibody molecules can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide comprising a nucleotide sequence encoding an antibody or antibody fragment (for example, a light chain or a heavy chain) is described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (for example, light chain or heavy chain) coding sequences and appropriate transcription and translation control signals. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. A replicable vector is also provided, which includes a nucleotide sequence encoding the antibody molecule, antibody heavy or light chain, antibody or fragment thereof heavy chain or light chain variable domain or heavy chain or light chain CDR, which can be It is operatively linked to a promoter. Such vectors can, for example, include nucleotide sequences encoding the constant regions of antibody molecules (see, for example, International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody It is colonized into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy chain and the light chain.

The expression vector can be transferred to cells (for example, host cells) by conventional techniques, and then can be cultured by conventional techniques The resulting cells are used to produce the antibodies described herein (e.g., antibodies including the CDR of pab1949 or pab2044) or fragments thereof. Therefore, a host cell is provided herein, which comprises a polynucleoside encoding an antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044) or a fragment thereof (for example, a heavy chain or light chain, or a fragment thereof) The acid is operably linked to the promoter to express such sequences in the host cell. In some embodiments, in order to express a double-chain antibody, a vector that separately encodes both the heavy chain and the light chain can be co-expressed in the host cell to express the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector that includes polynucleotides encoding both the heavy and light chains, or fragments thereof, of the antibodies described herein (e.g., antibodies that include the CDRs of pab1949 or pab2044). In a specific embodiment, the host cell contains two different vectors. The first vector includes the heavy chain or heavy chain variable region encoding the antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044), or a fragment thereof The polynucleotide, and the second vector include polynucleotides encoding the light chain or light chain variable region, or fragments thereof, of the antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044). In other embodiments, the first host cell includes a first vector that includes a heavy chain or heavy chain variable region encoding the antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044), or a fragment thereof And the second host cell includes a second vector that includes a polynucleotide encoding the light chain or light chain variable region of the antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044) . In a specific embodiment, the heavy chain/heavy chain variable region expressed by the first cell The light chain/light chain variable region of the second cell is combined to form an anti-OX40 antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044). In certain embodiments, a host cell population is provided herein, which includes such a first host cell and such a second host cell.

In a specific embodiment, a vector population is provided here, which comprises a first vector and a second vector, the first vector comprising an anti-OX40 antibody (for example, an antibody comprising the CDR of pab1949 or pab2044) described herein. The polynucleotide of the chain/light chain variable region, and the second vector includes the polynucleotide encoding the heavy chain/heavy chain variable region of the anti-OX40 antibody described herein (for example, an antibody including the CDR of pab1949 or pab2044) .

A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (e.g., antibodies that include the CDRs of pab1949 or pab2044) (see, e.g., U.S. Patent No. 5,807,715). This type of host expression system represents a vehicle by which the coding sequence of interest can be produced and subsequently purified, and also represents a cell that can express the antibody molecules described herein in situ when transformed or transfected with the appropriate nucleotide coding sequence. Such systems include, but are not limited to, microorganisms, such as bacteria (such as E. coli and Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; recombinant yeast expression vectors containing antibody coding sequences Transformed yeast (such as yeast (Saccharomyces), Pichia (Pichia)); insect cell systems infected with recombinant virus expression vectors (such as baculovirus) containing antibody coding sequences; using recombinant virus expression vectors (such as cauliflower flower leaf) Virus, CaMV; Tobacco mosaic virus, TMV) infected or transformed with recombinant plasmid expression vector containing antibody coding sequence (such as Ti plasmid) plant cell system (for example, green algae such as Chlamydomonas reinhardtii); The genome of animal cells (e.g. metallothionein promoter) or the mammalian cell system (e.g. COS( For example, COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, Hela, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM , BSC1, BSC40, YB/20 and BMT10 cells). In a specific embodiment, the cell line CHO cells used to express the antibodies described herein (for example, antibodies comprising the CDRs of any of the antibodies pab1949 or pab2044), such as CHO cells from the CHO GS System ^TM (Lonza) cell. In a specific embodiment, the cell line used to express the antibodies described herein is a human cell, for example, a human cell line. In a specific embodiment, the mammalian expression vector is pOptiVEC ^™ or pcDNA3.3. In a specific embodiment, bacterial cells (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells) are used for expressing recombinant antibody molecules, especially for expressing whole recombinant antibody molecules. For example, mammalian cells (such as Chinese hamster ovary (CHO) cells) combined with vectors (such as the main mid-early gene promoter elements from human cytomegalovirus) are effective expression systems for antibodies (Foerking (Foecking) MK and Huo Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al. (1990) Biotechnology 8: 662-667). In some embodiments, the antibody system described herein is produced by CHO cells or NSO cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antibody described herein that immunospecifically binds to OX40 (e.g., human OX40) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In a bacterial system, depending on the intended use of the expressed antibody molecule, many expression vectors can be advantageously selected. For example, when a large amount of such antibodies are to be produced for the production of pharmaceutical compositions of antibody molecules, it may be desirable to guide the high-level expression of fusion protein products that are easy to purify. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U and Mueller-Hill B, (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be Separately ligated into the vector in frame with the lac Z coding region, so that a fusion protein is produced; pIN vector (Inouye S and Inouye M (1985) Nuc Acids Res 13:3101-3109; Van He Van Heeke G and Schuster SM (1989) J Biol Chem 24:5503-5509); etc. For example, the pGEX vector can also be used to express an exogenous polypeptide as a fusion protein with glutaminylsulfur 5-transferase (GST). Generally speaking, this type of fusion protein is soluble and can be easily removed from the lysed cells by adsorbing and binding to the matrix glutamine sulfide-agarose beads, followed by elution in the presence of free glutamine sulfide purification. These pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that they can be released from the GST portion The target gene product of selection.

In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned separately into non-essential regions of the virus (for example, the polyhedrin gene) and placed under the control of an AcNPV promoter (for example, the polyhedrin promoter).

In mammalian host cells, many virus-based expression systems are available. In the case of using adenovirus as the expression vector, the coding sequence of the antibody of interest can be linked to the adenovirus transcription/translation regulatory complex, such as the late promoter and the tripartite leader sequence. Then, the chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (for example, region E1 or E3) will result in a recombinant virus that can survive and express antibody molecules in the infected host (for example, see Logan J and Shenk T (1984) Proceedings of the National Academy of Sciences (PNAS) 81: 3655-3659). A specific initiation signal is also required for efficient translation of the inserted antibody coding sequence. These signals include the ATG start codon and adjacent sequences. In addition, the initiation codon must be synchronized with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can have multiple sources, natural and synthetic. The efficiency of performance can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Enzymatic Method Methods Enzymol 153:516-544).

In addition, a host cell strain can be selected that regulates the performance of the inserted sequence, or modifies and processes the gene product in a specific way desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important to the function of the protein. Different host cells have characteristics and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed heterologous protein. For this purpose, eukaryotic host cells that process cell machinery can be used to properly process primary transcripts, glycosylation, and phosphorylation of gene products. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (will not endogenously produce any immunoglobulin Murine myeloma cell line of protein chain), CRL7O3O, COS (for example, COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB/20, BMT10 and HsS78BsT cells. In certain embodiments, the anti-OX40 antibodies described herein (e.g., antibodies that include the CDRs of pab1949 or pab2044) are produced in mammalian cells (e.g., CHO cells).

In a specific embodiment, the antibodies described herein have low fucose content or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, antibodies can be expressed in cells that are defective in fucosylation or lack the ability to fucosylate. In a specific example, a cell line with two alleles knocking out α1,6-fucosyltransferase can be used to produce antibodies with low fucose content. The Potelligent ^® system (Lonza) is an example of such a system and can be used to produce antibodies with low fucose content.

For long-term, high-yield production of recombinant protein, stable expression cells can be produced. For example, cell lines that stably express the anti-OX40 antibodies described herein (e.g., antibodies that include the CDRs of pab1949 or pab2044) can be engineered. In a specific embodiment, the cells provided herein stably exhibit light chain/light chain variable domains and heavy chain/heavy chain variables associated with the formation of antibodies described herein (for example, antibodies comprising the CDRs of pab1949 or pab2044) area.

In some aspects, instead of using expression vectors containing viral origins of replication, DNA and DNA controlled by appropriate expression control elements (for example, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) can be used. The selection marker transforms the host cell. After introducing the exogenous DNA/polynucleotide, the engineered cells can be allowed to grow in enriched media for 1 to 2 days, and then switched to selective media. The selection marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form lesions, which can then be colonized and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the anti-OX40 antibodies or fragments thereof described herein. Such engineered cell lines are particularly suitable for screening and evaluating components that directly or indirectly interact with the antibody molecule.

Many selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1): 223-232), hypoxanthine Transphosphoribosylase (Szybalska EH and Szybalski W (1962) Proceedings of the National Academy of Sciences (PNAS) 48(12): 2026-2034) and adenine phosphoribosyl transfer The enzyme (Lowy I et al., (1980) Cell 22(3):817-823) gene can be used in tk-, hgprt- or aprt-cells, respectively. In addition, antimetabolites resistance can be used as a basis for selecting the following genes: dhfr , which confers resistance to methotrexate (Wigler M et al. (1980) Proceedings of the National Academy of Sciences (PNAS) 77 (6): 3567-3570; O'Hare K et al., (1981) Proceedings of the National Academy of Sciences 78:1527-1531); gpt , which confers resistance to mycophenolic acid (Mulligan ) RC and Berger (Berg) P (1981) Proceedings of the National Academy of Sciences 78(4): 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Wu (Wu) GY and Wu (Wu) CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mu Ligan RC (1993) Science 260: 926-932; and Morgan RA and Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel ) GJ and Felgner (Felgner) PL (1993) Trends Biotechnol 11(5): 211-215); and hygro, which confers resistance to hygromycin (Santerre RF etc. Human, (1984) Gene 30(1-3):147-156). The methods of recombinant DNA technology known in the art can be routinely applied to select the desired recombinant clones, and such methods are described in, for example, Ausubel FM et al. (Editor), Contemporary Molecular Biology Experiments Manual (Current Protocols in Molecular Biology), John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression: Laboratory Manual (Gene Transfer and Expression) , A Laboratory Manual), Stockton Press (Stockton Press), NY (1990); and in Chapters 12 and 13, Dracopoli NC et al. (editors), Current Protocols in Human Genetics (Current Protocols in Human Genetics), John & Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150:1-14, which is incorporated in its entirety by reference this.

The expression level of antibody molecules can be increased by vector amplification (for a review, see Bebbington CR and Hentschel CCG, using a vector based on gene amplification to generate DNA clones in mammalian cells). The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987). When the marker in the vector system expressing the antibody can be amplified, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse GF et al., (1983), Mol Cell Biol 3:257-66).

The host cell can be co-transfected with two or more of the expression vectors described herein (the first vector encodes a heavy chain-derived polypeptide and the second vector encodes a light chain-derived polypeptide). These two vectors can contain the same selection marker, which allows the heavy chain and light chain polypeptides to behave equally. The host cell can be co-transfected with different amounts of two or more expression vectors. For example, the host cell can be transfected with the first expression vector and the second expression vector in any of the following ratios: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1: 7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such cases, the light chain should be placed before the heavy chain to avoid excessive toxic free heavy chains (Proudfoot NJ (1986) Nature 322:562-565; and Köhler ) G (1980) Proceedings of the National Academy of Sciences (PNAS) 77: 2197-2199). The heavy and light chain coding sequences may include cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. The polycistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or genes in the range of 2 to 5, 5 to 10, or 10 to 20 /Nucleotide sequence. For example, a bicistronic nucleic acid construct may include a promoter, a first gene (e.g., an antibody heavy chain described herein), and a second gene and (e.g., an antibody light chain described herein) in the following order. In this expression vector, the transcription of the two genes can be driven by the promoter, and the translation of the mRNA from the first gene can be driven by the cap-dependent scanning mechanism. Drive, and the translation of mRNA from the second gene can be driven by a cap-independent mechanism (for example, by IRES).

Once the antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange , Affinity (especially by specific antigen affinity for protein A) and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard techniques used to purify proteins. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or other known in the art to facilitate purification.

In a specific embodiment, the antibodies described herein are isolated or purified. Generally, the isolated antibody system is substantially free of other antibodies with different antigenic specificities from the isolated antibody. For example, in one embodiment, the antibody formulation described herein is substantially free of cellular material and/or chemical precursors. The language "substantially free of cellular material" includes preparations of antibodies in which the antibody system is isolated from the cellular components of the cell, and the antibody is isolated or recombinantly produced from the cell. Therefore, an antibody that is substantially free of cellular material includes a heterologous protein having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (by dry weight) This is also referred to as a "contaminating protein") and/or antibody variants (for example, different post-translationally modified forms of antibodies) antibody preparations. When the antibody or fragment is produced recombinantly, it is also generally substantially free of culture medium, that is, culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When an antibody or fragment is produced by chemical synthesis, it usually contains essentially no chemical precursors or Other chemical agents, that is, separated from chemical precursors or other chemical agents involved in the synthesis of proteins. Therefore, preparations of such antibodies or fragments have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or ingredients other than the antibody or fragment of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

5.4 Pharmaceutical composition

A composition is provided here, which includes the physiologically acceptable carrier, excipient, or stabilizer of the antibody having the desired purity described herein (Remington's Pharmaceutical Sciences (1990) Mark Publishing Company (Mack Publishing Co.), Easton, Pennsylvania). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration used.

The pharmaceutical composition described herein can be used to enhance, induce or activate OX40 activity and treat disorders (such as cancer or infectious diseases). Examples of cancers that can be treated according to the methods described herein include, but are not limited to: B-cell lymphoma (e.g., B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin’s lymphoma, skin B-cell lymphoma, diffuse large B-cell lymphoma), basal cell carcinoma, bladder cancer, blastoma, brain metastases, breast cancer, Burkitt lymphoma, cancer (e.g., adenocarcinoma (e.g., adenocarcinoma at the junction of the stomach and esophagus) )), cervical cancer, colon cancer, colorectal cancer (colon cancer and rectal cancer), endometrial cancer, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, stomach and Cancer of the esophageal junction, gastrointestinal cancer, glioblastoma (e.g., glioblastoma multiforme (e.g., newly diagnosed or recurring)), glioma, head and neck cancer (e.g., head and neck Squamous cell carcinoma), liver metastases, Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, kidney cancer (for example, renal cell carcinoma and Wilms' tumor), laryngeal cancer, leukemia ( For example, chronic myeloid leukemia, hairy cell leukemia), liver cancer (e.g., hepatic carcinoma and liver tumors), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoblastic lymphoma , Lymphoma, mantle cell lymphoma, metastatic brain tumor, metastatic cancer, myeloma (e.g., multiple myeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer (E.g., pancreatic duct adenocarcinoma), prostate cancer (e.g., hormone-resistant (e.g., castration-refractory), metastatic, metastatic hormone-refractory (e.g., castration-refractory, non-androgen-dependent)), renal cell carcinoma (E.g., metastatic), salivary gland cancer, sarcoma (e.g., rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g., metastatic melanoma)), soft tissue sarcoma, solid tumor, squamous cell carcinoma, synovial sarcoma , Testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell cancer), uveal melanoma (for example, metastatic), verrucous carcinoma, Waldenstrom macroglobulinemia. In one embodiment, examples of cancers that can be treated according to the methods described herein include, but are not limited to: advanced, recurrent, or metastatic solid tumors, lymphomas (e.g., diffuse large B-cell lymphoma or Burke Special lymphoma), breast cancer, prostate cancer, head and neck cancer, colon Rectal cancer, colon cancer, melanoma (e.g., metastatic melanoma), endometrial cancer, renal cell carcinoma, renal clear cell carcinoma, lung cancer (e.g., non-small cell lung cancer or lung adenocarcinoma), ovarian cancer, gastric cancer, Bladder cancer, gastric cancer, uterine cancer, pheochromocytoma, metastatic cutaneous squamous cell carcinoma (e.g., in transplant patients), Merkel's cell carcinoma, cutaneous T-lymphoma, neuroendocrine tumors, bone-derived tumors (e.g., Osteosarcoma), hemangiopericytoma, tumors related to genetic syndromes (NF1 or VHL), chordoma, ependymoma, medulloblastoma, germ cell tumor, small bowel tumor, appendix cancer, and virus-related Tumor (for example, Kaposi's sarcoma). In one embodiment, the pharmaceutical composition described herein is used as a medicine or diagnostic agent. In one embodiment, the pharmaceutical composition including the agonistic antibody described herein is used in a method of treating cancer.

The pharmaceutical compositions described herein including the antagonist antibodies described herein can be used to reduce, inhibit, or inactivate OX40 activity and treat disorders (such as inflammatory or autoimmune diseases or disorders or infectious diseases). In one embodiment, the pharmaceutical composition including the antagonist antibody described herein is used in a method of treating inflammatory or autoimmune diseases or disorders or infectious diseases.

The pharmaceutical composition described herein including the antagonist antibody described herein can be used to reduce, inactivate, or inhibit the activity of OX40 and treat a disorder selected from the group consisting of: infection (viral, bacterial (Sexual, fungal, and parasites), infection-related endotoxin shock, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer’s disease, inflammatory bowel disease, gram Ron Diseases, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, Tibetan disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, uveitis , Autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems (such as multiple sclerosis, lupus (such as systemic lupus erythematosus)), and Ge-Bar syndrome, dermatitis, abnormalities Positional dermatitis, autoimmune hepatitis, fibrous alveolitis, Gray's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary liver Scleroderma, sarcoidosis, scleroderma, Wagner's granulation disease, pancreatitis, trauma (surgery), graft versus host disease, transplant rejection, heart disease (i.e. cardiovascular disease) (including ischemic disease ( Such as myocardial infarction and arteriosclerosis), intravascular blood coagulation, bone resorption, osteoporosis, osteoarthritis, root periostitis, hypochlorhydia, optic neuromyelitis, celiac disease, connective tissue disease (e.g., lupus ), post-infection inflammatory diseases (eg Geba's syndrome), and tumor-associated syndrome.

The compositions for in vivo administration may be sterile. This can be easily achieved by filtering through, for example, a sterile filter membrane.

5.5 Uses and methods

5.5.1 Therapeutic uses and methods

In one aspect, a method for modulating one or more immune functions or responses of a subject is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof described herein to a subject in need thereof. In a specific aspect, a method for initiating, enhancing or inducing one or more immune functions or responses in a subject is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof . In a specific embodiment, a method for preventing and/or treating a disease in which it is desired to initiate or enhance one or more immune functions or responses is presented here, the method comprising administering to a subject in need thereof The described anti-OX40 antibody or composition thereof. In one embodiment, a method for the treatment of infectious diseases is presented here, and the method includes administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In one embodiment, a method for treating cancer is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. Cancer can be selected from the group consisting of: melanoma, kidney cancer, and prostate cancer. Cancer can be selected from the group consisting of: melanoma, kidney cancer, prostate cancer, colon cancer, and lung cancer. In a certain embodiment, a method for treating melanoma is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In a certain embodiment, a method for treating kidney cancer is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In a certain embodiment, a method for treating prostate cancer is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In certain embodiments, a method for treating colon cancer is presented herein, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In some embodiments, a method for the treatment of lung cancer is presented here, the method comprising Of subjects were given anti-OX40 antibody or its composition. In certain embodiments, a method for the treatment of non-small cell lung cancer (NSCLC) is presented here, the method comprising administering an anti-OX40 antibody or a composition thereof to a subject in need thereof. In one example, the method further includes administering a checkpoint targeting agent to the subject. In one example, the checkpoint targeting agent is selected from the group consisting of: antagonist anti-PD-1 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-CTLA-4 Antibodies, antagonist anti-TIM-3 antibodies, antagonist anti-LAG-3 antibodies, antagonist anti-CEACAM1 antibodies, agonist anti-GITR antibodies, agonist anti-CD137 antibodies, and agonist anti-OX40 antibodies. In certain embodiments, the checkpoint targeting agent is an antagonist anti-PD-1 antibody. In certain embodiments, the checkpoint targeting agent is an antagonist anti-PD-L1 antibody. In certain embodiments, the checkpoint targeting agent is an agonist anti-GITR antibody. In certain embodiments, the checkpoint targeting agent is an agonist anti-CD137 antibody.

In certain embodiments, anti-PD-1 antibodies are used in the methods disclosed herein. In some embodiments, the anti-PD-1 antibody system Nivolumab (Nivolumab), also known as BMS-936558 or MDX1106, was developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-1 antibody system Pembrolizumab (Pembrolizumab), also known as Lambrolizumab (Lambrolizumab) or MK-3475, was developed by Merck & Co. In some embodiments, the anti-PD-1 antibody system Pidilizumab (Pidilizumab), also known as CT-011, was developed by CureTech. In some embodiments, the anti-PD-1 antibody system MEDI0680, also known as AMP-514, is produced by the Medical Immunization Company (Medimmune) developed. In some embodiments, the anti-PD-1 antibody system is PDR001 developed by Novartis Pharmaceuticals. In some embodiments, the anti-PD-1 antibody system is REGN2810 developed by Regeneron Pharmaceuticals. In some embodiments, the anti-PD-1 antibody system is PF-06801591 developed by Pfizer. In some embodiments, the anti-PD-1 antibody system is BGB-A317 developed by BeiGene. In some embodiments, the anti-PD-1 antibody system is TSR-042 developed by AnaptysBio and Tesaro. In some embodiments, the anti-PD-1 antibody system is SHR-1210 developed by Hengrui.

Other non-limiting examples of anti-PD-1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes. : US Patent No. 6,808,710; US Patent No. 7,332,582; US Patent No. 7,488,802; US Patent No. 8,008,449; US Patent No. 8,114,845; US Patent No. 8,168,757; US Patent No. 8,354,509; US Patent No. 8,686,119; US US Patent No. 8,735,553; US Patent No. 8,747,847; US Patent No. 8,779,105; US Patent No. 8,927,697; US Patent No. 8,993,731; US Patent No. 9,102,727; US Patent No. 9,205,148; US Publication No. US 2013/0202623 A1 ; US Open Case No. US 2013/0291136 A1; US Open Case No. US 2014/0044738 A1; US Open Case No. US 2014/0356363 A1; U.S. Publication No. US 2016/0075783 A1; and PCT Publication No. WO 2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT Publication No. WO 2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCT Publication No. WO 2014/206107 A1; PCT Publication No. WO 2015/058573 A1; PCT Publication No. WO 2015/085847 A1; PCT Publication No. WO 2015/200119 A1; PCT Publication No. WO 2016/015685 A1; and PCT publication number WO 2016/020856 A1.

In certain embodiments, anti-PD-L1 antibodies are used in the methods disclosed herein. In some embodiments, the anti-PD-L1 antibody system is atezolizumab developed by Genentech. In some embodiments, the anti-PD-L1 antibody system is durvalumab developed by AstraZeneca, Celgene, and Medical Immunology. In some embodiments, the anti-PD-L1 antibody system avelumab, also known as MSB0010718C, was developed by Merck Serono and Pfizer. In some embodiments, the anti-PD-L1 antibody system is MDX-1105 developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-L1 antibody system is AMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that can be used in the therapeutic methods disclosed herein are disclosed in the following patents and patent applications, which are incorporated by reference in their entirety by reference for all purposes: United States of America Patent case number 7,943,743; US patent case number 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. Publication No. US 2003/0232323 A1; US Open Case No. US 2013/0323249 A1; US Open Case No. US 2014/0341917 A1; US Open Case No. US 2014/0044738 A1; US Open Case No. US 2015/0203580 A1; US Open Case No. US 2015/0225483 A1; US Publication No. US 2015/0346208 A1; US Publication No. US 2015/0355184 A1; and PCT Publication No. WO 2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT Publication No. WO 2014/055897 A2 ; PCT Publication No. WO 2015/061668 A1; PCT Publication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163 A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO 2016/030350 A1.

In one embodiment, a method for treating a cancer selected from the group consisting of B-cell lymphoma (e.g., B-cell chronic lymphocytic leukemia, B-cell non-Hojie King's lymphoma, skin B-cell lymphoma, diffuse large B-cell lymphoma), basal cell carcinoma, bladder cancer, blastoma, brain metastases, breast cancer, Burkitt lymphoma, cancer (e.g. , Adenocarcinoma (for example, adenocarcinoma at the junction of the stomach and esophagus), cervical cancer, colon cancer, colorectal cancer (colon and rectal cancer), endometrial cancer, esophageal cancer, Ewing’s sarcoma, follicular Lymphoma, gastric cancer, cancer at the junction of the stomach and esophagus, gastrointestinal cancer, glioblastoma (e.g., glioblastoma multiforme) Blastoma (e.g., newly diagnosed or recurring), glioma, head and neck cancer (e.g., head and neck squamous cell carcinoma), liver metastases, Hodgkin’s lymphoma and non-Hodgkin’s lymphoma , Kidney cancer (e.g., renal cell carcinoma and Wilms' tumor), laryngeal cancer, leukemia (e.g., chronic myeloid leukemia, hairy cell leukemia), liver cancer (e.g., liver cancer ( hepatic carcinoma) and liver tumors), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoblastic lymphoma, lymphoma, mantle cell lymphoma, metastatic brain tumor, metastatic cancer, myeloma (e.g., Multiple myeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatic duct adenocarcinoma), prostate cancer (e.g., hormone-resistant (e.g., castration refractory) ), metastatic, metastatic hormone refractory (e.g., castration refractory, non-androgen-dependent), renal cell carcinoma (e.g., metastatic), salivary gland cancer, sarcoma (e.g., rhabdomyosarcoma), skin cancer (e.g., Melanoma (e.g., metastatic melanoma), soft tissue sarcoma, solid tumor, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell cancer), uveal melanoma ( For example, metastatic), verrucous carcinoma, Waldenstrom macroglobulinemia. In a certain embodiment, a method for treating cancer selected from the group consisting of advanced, recurrent, or metastatic solid tumors, lymphoma (e.g., diffuse Large B-cell lymphoma or Burkitt’s lymphoma), breast cancer, prostate cancer, head and neck cancer, colorectal cancer, colon cancer, melanoma (e.g., metastatic melanoma), endometrial cancer, renal cell carcinoma, Renal clear cell carcinoma, lung cancer (for example, non-small cell lung cancer or lung adenocarcinoma), ovarian cancer, gastric cancer, bladder cancer, gastric cancer, uterine cancer, pheochromocytoma, metastatic skin squamous cell carcinoma (for example, in transplant patients Middle), Merkel's cell carcinoma, cutaneous T-lymphoma, neuroendocrine tumors, bone-derived tumors (e.g., osteosarcoma), hemangiopericytoma, tumors related to genetic syndromes (NF1 or VHL), chordomas, ventricular Ependymoma, medulloblastoma, germ cell tumor, small intestine tumor, appendix cancer, and virus-related tumors (e.g., Kaposi's sarcoma).

In another embodiment, the anti-OX40 antibody is administered to a patient diagnosed with cancer to increase the proliferation of one or more immune cell populations (for example, T cell effector cells (such as CD4 ⁺ and CD8 ⁺ T cells) and/ Or effector function.

In a specific embodiment, the anti-OX40 antibody described herein initiates or enhances or induces one or more immune functions or responses of the subject relative to the immune function of a subject to which the anti-OX40 antibody described herein is not administered At least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20% or at least 10% or between 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95% Within the scope of this, well-known assays in the art (e.g., ELISPOT, ELISA) and cell proliferation assays are used. In a specific embodiment, the immune function is cytokine production (eg, IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13 production). In another implementation In the method, the proliferation/expansion of T cells of immune function can be measured by, for example, flow cytometry to detect the number of cells expressing T cell markers (for example, CD3, CD4, or CD8). In another embodiment, the immune function is related to antibody production, which can be measured by, for example, ELISA. In some embodiments, the immune function system effector function can be determined by, for example, cytotoxicity analysis or other analysis well-known in the art. In another embodiment, the immune function is a Th1 response. In another embodiment, the immune function is a Th2 response. In another embodiment, the immune function is a memory response.

In specific embodiments, non-limiting examples of immune functions that can be enhanced or induced by anti-OX40 antibodies are: effector lymphocyte proliferation/expansion (for example, increase in the number of effector T lymphocytes), and suppression of effector lymphocytes (For example, effector T lymphocytes) apoptosis. In a specific embodiment, the immune function enhanced or induced by the anti-OX40 antibodies described herein: CD4 ⁺ T cells (for example, Th1 and Th2 helper T cells), CD8 ⁺ T cells (for example, cytotoxic T lymphocytes) , Α/β T cells, and γ/δ T cells), B cells (for example, plasma cells), memory T cells, memory B cells, tumor-resident T cells, CD122 ⁺ T cells, natural killer (NK) cells) , Macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils or polymorphonuclear leukocyte proliferation/number expansion or activation. In one embodiment, the anti-OX40 antibodies described herein initiate or enhance the proliferation/expansion or number of lymphocyte precursors. In some embodiments, the anti-OX40 antibody described herein will reduce CD4 ⁺ T cells (e.g., Th1 And Th2 helper T cells), CD8 ⁺ T cells (for example, cytotoxic T lymphocytes, α/β T cells, and γ/δ T cells), B cells (for example, plasma cells), memory T cells, memory B cells , Tumor-resident T cell (tumor-resident T cell), CD122 ⁺ T cell, natural killer (NK) cell), macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils The number of granulocytes or polymorphonuclear leukocytes increased by approximately at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%, or between 10% and 25%, 25% and 50%, In the range of 50% to 75%, or 75% to 95%.

In some embodiments, the anti-OX40 antibodies described herein are combined with one or more immunomodulatory enzymes (such as IDO (indoleamine-2,3-dioxygenase) and TDO (tryptophan 2,3- The compound combination of dioxygenase) is administered to the subject. In a specific embodiment, such compound is selected from the group consisting of: epacadostat (Incyte Corp (Incyte Corp) )), F001287 (Flexus Biosciences), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound series Epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is Epacadostat. (indoximod). In another embodiment, the compound is NLG919.

In some embodiments, the anti-OX40 antibody described herein is administered to the subject in combination with a vaccine.

In some embodiments, the anti-OX40 antibody described herein is administered to the subject in combination with an anti-CD137 antibody, rituximab, cyclophosphamide, chemotherapy, or radiation therapy.

In some embodiments, the anti-OX40 antibody described herein is administered to the subject in combination with a heat shock protein-based tumor vaccine or a heat shock protein-based pathogen vaccine. In a specific embodiment, an anti-OX40 antibody is administered to the subject in combination with a heat shock protein-based tumor vaccine. Heat shock proteins (HSP) are a family of highly conserved proteins that are ubiquitous in all species. As a result of heat shock or other forms of stress (including exposure to toxins, oxidative stress, or glucose deprivation), their performance can be strongly induced to higher levels. According to molecular weight, it is classified into five families: HSP-110, -90, -70, -60 and -28. HSP delivers immunogenic peptides via the cross-presentation pathway of antigen presenting cells (APC), such as macrophages and dendritic cells (DC), which leads to T cell activation. HSP functions as a protein chaperone carrier for tumor-associated antigen peptides that can induce tumor-specific immune complexes. Once released from dead tumor cells, the HSP-antigen complex is absorbed by antigen presenting cells (APC), where the antigen is processed into peptides that bind to MHC class I and class II molecules, resulting in the activation of anti-tumor CD8+ and CD4+ T cells . The immunity induced by the HSP complex derived from the tumor preparation is specific to the cancer manifested by each subject Unique antigen peptide antibody library.

Heat shock protein peptide complex (HSPPC) is a protein peptide complex composed of heat shock protein non-covalently complexed with antigen peptides. HSPPC elicits both innate and acquired immune responses. In a specific embodiment, one or more antigenic peptides exhibit antigenicity against the cancer being treated. HSPPC can be effectively captured by APC via membrane receptors (mainly CD91) or by binding to Toll-like receptors. The internalization of HSPPC leads to the functional maturation of APCs with chemokines and cytokines, leading to the activation of natural killer cells (NK), monocytes, and Th1 and Th-2 mediated immune responses. In some embodiments, the HSPPC used in the methods disclosed herein includes one or more heat shock proteins from the hsp60, hsp70, or hsp90 family of stress proteins complexed with antigen peptides. In some embodiments, HSPPC includes hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or a combination of two or more thereof.

In a specific embodiment, an anti-OX40 antibody in combination with a heat shock protein peptide complex (HSPPC) (eg, heat shock protein peptide complex-96 (HSPPC-96)) is administered to a subject to treat cancer. HSPPC-96 includes 96kDa heat shock protein (Hsp), gp96, which is compounded with antigen peptide. HSPPC-96 is a cancer immunotherapy made from the subject's tumor and contains the antigen "fingerprint" of cancer. In some embodiments, the fingerprint contains unique antigens that are only present in specific cancer cells of a specific subject, and the injection of the vaccine is intended to stimulate the subject’s immune system to identify and attack any specific cancer fingerprint. cell.

In some embodiments, HSPPC (e.g., HSPPC-96) is produced from tumor tissue in the subject. In a specific embodiment, HSPPC (e.g., HSPPC-96) is produced from a tumor of the cancer type being treated or its metastasis. In another specific embodiment, the HSPPC (e.g., HSPPC-96) is autologous to the subject being treated. In some embodiments, the tumor tissue is a non-necrotic tumor tissue. In some embodiments, at least 1 gram (eg, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 grams) of non-necrotic tumor tissue is used To produce a vaccine program. In some embodiments, after surgical resection, the non-necrotic tumor tissue is frozen before being used in vaccine preparation. In some embodiments, HSPPC (eg, HSPPC-96) is separated from tumor tissues by purification technology, filtered, and prepared into an injectable vaccine. In some embodiments, 6-12 doses of HSPPC (e.g., HSPPC-96) are administered to the subject. In such embodiments, the HSPPC (e.g., HSPPC-96) dose may be given in 4 doses initially per week, and then 2-8 additional doses biweekly.

Other examples of HSPPC that can be used according to the methods described herein are disclosed in the following patents and patent applications, which are incorporated by reference in their entirety for all purposes: U.S. Patent Nos. 6,391,306, 6,383,492 , 6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659.

In some embodiments, the present invention relates to the use of the antibody or pharmaceutical composition of the present invention as a medicine. In some aspects, the present invention relates to the antibody or pharmaceutical composition of the present invention for use in a method of treating cancer. exist In some aspects, the present invention relates to the antibody or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or pharmaceutical composition of the present invention. In some aspects, the present invention relates to (a) the antibody or pharmaceutical composition of the present invention and (b) a checkpoint targeting agent for use as a medicine. In some aspects, the present invention relates to (a) an antibody or pharmaceutical composition of the present invention and (b) a checkpoint targeting agent for use in a method for treating cancer. In some aspects, the present invention relates to a composition, kit, or multi-part kit, which includes (a) the antibody or pharmaceutical composition of the invention and (b) a checkpoint targeting agent. In one aspect, the invention relates to (a) the antibody or pharmaceutical composition of the invention and (b) an IDO inhibitor for use as a medicine. In some aspects, the invention relates to (a) an antibody or pharmaceutical composition of the invention and (b) an IDO inhibitor for use in a method of treating cancer. In some aspects, the present invention relates to a composition, kit, or multi-part kit, which includes (a) an antibody or pharmaceutical composition of the invention and (b) an IDO inhibitor. In some aspects, the invention relates to (a) the antibody or pharmaceutical composition of the invention and (b) a vaccine for use as a medicine. In some aspects, the invention relates to (a) the antibody or pharmaceutical composition of the invention and (b) a vaccine for use in a method of treating cancer. In some aspects, the present invention relates to a composition, kit, or multi-part kit, which includes (a) the antibody or pharmaceutical composition of the invention and (b) a vaccine. In a preferred embodiment where the antibody or pharmaceutical composition is used in a method of treating cancer, the anti-system agonistic.

In one aspect, the method for modulating one or more immune functions or responses of a subject as presented herein is for inactivating, reducing or A method of inhibiting one or more immune functions or responses of a subject, the method comprising administering an anti-OX40 antagonist antibody or a composition thereof to a subject in need thereof. In a specific embodiment, a method for preventing and/or treating a disease in which it is desired to inactivate, reduce, or suppress one or more immune functions or responses is presented here, and the method comprises providing a subject in need thereof The anti-OX40 antagonist antibody described herein or a composition thereof is administered. In a certain embodiment, a method for the treatment of autoimmune or inflammatory diseases or disorders is presented here, the method comprising administering to a subject in need thereof an effective amount of an anti-OX40 antagonist antibody or a composition thereof . In some embodiments, the subject is human. In some embodiments, the disease or disorder is selected from the group consisting of: infection (viral, bacterial, fungal, and parasite), infection-related endotoxin shock, arthritis, rheumatoid arthritis , Asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer’s disease, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, Peyronie’s disease, celiac disease, gallbladder disease, Tibetan disease, peritonitis, Psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, uveitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems (E.g. multiple sclerosis, lupus (e.g. systemic lupus erythematosus)) and Guad-Bar syndrome, dermatitis, atopic dermatitis, autoimmune hepatitis, fibrous alveolitis, Gray's disease, IgA nephropathy, Idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wagner's granulomatosis, pancreatitis, trauma (surgery), Graft versus host disease, transplant rejection, heart disease (i.e. cardiovascular disease) (including Including ischemic diseases (such as myocardial infarction and arteriosclerosis), intravascular blood coagulation, bone resorption, osteoporosis, osteoarthritis, root periostitis, hypochlorhydia, optic neuromyelitis, celiac disease, connective Tissue disease (e.g., lupus), post-infection inflammatory disease (e.g., Geba's syndrome), and tumor-associated syndrome. In certain embodiments, the disease or disorder is selected from the group consisting of transplant rejection, vasculitis, asthma, rheumatoid arthritis, dermatitis, inflammatory bowel disease, uveitis, and lupus. In certain embodiments, any of the methods herein (for example, a method for treating an infectious disease, or a method for treating an autoimmune or inflammatory disease or disorder) includes administering to a subject such as The antibodies and checkpoint targeting agents described here. In certain embodiments, the checkpoint targeting agent is an antibody (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody, anti-LAG- 3 Antibody, anti-CEACAM1 antibody, anti-GITR antibody, anti-CD137 antibody, or anti-OX40 antibody). In certain embodiments, the checkpoint targeting agent is an antagonist or agonist antibody. In some embodiments, the checkpoint targeting agent is an anti-PD-1 antibody. In certain embodiments, the checkpoint targeting agent is an anti-GITR antibody. In certain embodiments, the checkpoint targeting agent is an anti-CD137 antibody.

In another embodiment, an anti-OX40 antagonist antibody is administered to a patient diagnosed with an autoimmune or inflammatory disease or disorder to reduce one or more immune cell populations (e.g., T cell effector cells (such as CD4 ⁺ and CD8 ⁺ T cell) proliferation and/or effector function.

In a specific embodiment, relative to not given here The described anti-OX40 antagonist antibody of the subject’s immune function, the anti-OX40 antagonist antibody described herein inactivates or reduces or inhibits one or more of the subject’s immune function or response by at least 99%, at least 98%, At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30 %, at least 25%, at least 20%, or at least 10%, or within the range of 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95%, using well known in the art Assays (e.g. ELISPOT, ELISA) and cell proliferation assays. In a specific embodiment, the immune function is cytokine production (eg, IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13 production). In another embodiment, the proliferation/expansion of T cells of immune function can be measured by, for example, flow cytometry to detect the number of cells expressing T cell markers (eg, CD3, CD4, or CD8). In another embodiment, the immune function is related to antibody production, which can be measured by, for example, ELISA. In some embodiments, the immune function system effector function can be determined by, for example, cytotoxicity analysis or other analysis well-known in the art. In another embodiment, the immune function is a Th1 response. In another embodiment, the immune function is a Th2 response. In another embodiment, the immune function is a memory response.

In specific embodiments, non-limiting examples of immune functions that can be reduced or suppressed by anti-OX40 antagonist antibodies are: effector lymphocyte proliferation/expansion (for example, reduction in the number of effector T lymphocytes), and stimulatory effects Apoptosis of lymphocytes (for example, effector T lymphocytes). In a specific embodiment, the immune function reduced or suppressed by the anti-OX40 antagonist antibodies described herein: CD4 ⁺ T cells (for example, Th1 and Th2 helper T cells), CD8 ⁺ T cells (for example, cytotoxic T cells) Lymphocytes, α/β T cells, and γ/δ T cells), B cells (for example, plasma cells), memory T cells, memory B cells, tumor-resident T cells, CD122 ⁺ T Proliferation/number of cells, natural killer (NK) cells), macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils, or polymorphonucleated leukocytes Amplify or activate. In one embodiment, the anti-OX40 antagonist antibodies described herein inactivate or reduce or inhibit the proliferation/expansion or number of lymphocyte precursors. In some embodiments, relative to a negative control (e.g., the number of corresponding cells not treated, cultured, or contacted with the anti-OX40 antagonist antibody described herein), the anti-OX40 antagonist antibody described herein reduces CD4 ⁺ T cells (For example, Th1 and Th2 helper T cells), CD8 ⁺ T cells (for example, cytotoxic T lymphocytes, α/β T cells, and γ/δ T cells), B cells (for example, plasma cells), memory T cells , Memory B cells, tumor-resident T cells, CD122 ⁺ T cells, natural killer (NK) cells), macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils or The number of polymorphonucleated white blood cells is reduced by approximately at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, At least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%, or between 10% to 25%, 25% to 50%, 50% to 75%, or the range between 75% and 95%.

In some aspects, the present invention relates to the antibody or pharmaceutical composition of the present invention for use in a method of treating autoimmune or inflammatory diseases or disorders. In one aspect, the present invention relates to the antibody or pharmaceutical composition of the present invention for use in a method of treating infectious diseases. In a preferred embodiment of the antibody or pharmaceutical composition for use in a method of treating autoimmune or inflammatory diseases or disorders, or infectious diseases, the anti-system antagonistic.

5.5.1.1 Route of administration and dosage

The antibodies or compositions described herein can be delivered to a subject by a variety of routes, such as parenteral, subcutaneous, intravenous, intradermal, transdermal, intranasal, intratumoral, and administration to tumor-draining lymph nodes. In one embodiment, the antibody or composition is administered by intravenous or intratumoral route.

The amount of antibody or composition that will be effective in treating and/or preventing the disorder will depend on the nature of the disease and can be determined by standard clinical techniques.

The precise dosage used in the composition will also depend on the route of administration, and the severity of the disease, and should be determined based on the judgment of the practitioner and the conditions of each subject. For example, the effective dose may also vary depending on the method of administration, the target site, the physiological state of the patient (including age, weight, and health), whether the patient is a human or animal, and whether other drugs or treatments given are prophylactic or therapeutic. . Generally, the patient is a human, but non-human mammals including transgenic mammals can also be treated. The therapeutic dose can be optimized and titrated to optimize safety and efficacy.

In certain embodiments, in vitro assays are used to help identify optimal dosage ranges. The effective dose can be extrapolated from a dose response curve derived from an in vitro or animal model test system.

Generally, due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and lower frequency administration are usually possible.

5.5.2 Testing and diagnostic purposes

The anti-OX40 antibodies described here (see, for example, section 5.2) can be used to analyze OX40 protein levels in biological samples, using typical immunohistological methods known to those skilled in the art, including immunoassays, such as enzyme-linked immunosorbent assays (ELISA), immunoprecipitation or western blotting method. Suitable antibody assay tags are known in the art and include enzyme tags (e.g., glucose oxidase); radioisotopes (e.g., iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium) ( ³ H), indium ( ¹²¹ In) and ^{T (99} Tc)); luminescent labels (such as luminol); and fluorescent labels (such as luciferin and rhodamine and biotin). Such tags can be used to label the antibodies described herein. Alternatively, a second antibody that recognizes the anti-OX40 antibody described herein can be labeled and used in combination with the anti-OX40 antibody to detect OX40 protein levels.

Determining the expression level of OX40 protein is intended to include direct (for example, by determining or estimating absolute protein levels) or relative (for example, by comparison with disease-related protein levels in the second biological sample) qualitative Or quantitatively measure or estimate the level of OX40 protein. Measurable or Estimate and compare the OX40 polypeptide performance level in the first biological sample with the standard OX40 protein level. The standard is taken from the second biological sample obtained from individuals without the disease or from a population of individuals without the disease The average level is ok. As will be understood in the art, once the "standard" OX40 polypeptide level is known, it can be reused as a standard for comparison.

As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells that may exhibit OX40. Methods for obtaining biopsies and body fluids from animals (e.g., humans) are well known in the art. The biological sample includes peripheral blood mononuclear cells.

The anti-OX40 antibodies described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications that are well known and standard by skilled artisans and are based on this specification. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and/or immune response can be used for prediction, diagnosis and monitoring to evaluate patient samples, including known or suspected immune system -Those samples that are dysfunctional or about the expected or desired immune system response, antigen response or vaccine responder. The assessment and evaluation of immune system status and/or immune response can also be used to determine the suitability of patients for drug clinical trials or for the administration of specific chemotherapeutic agents or antibodies (including combinations thereof) relative to different agents or antibodies. This type of prognosis, diagnosis, and monitoring evaluation is already in practice, using antibodies against the HER2 protein in breast cancer (HercepTest ^™ , Dako), where the assay uses Herceptin® and is also used to evaluate patients with antibody therapy. In vivo applications include targeted cell therapy and immune system modulation and immune response radiography.

In one embodiment, anti-OX40 antibodies can be used in immunohistochemistry of biopsy samples.

In another embodiment, the anti-OX40 antibody can be used to detect the level of OX40 or the level of cells containing OX40 on its membrane surface, which can then be correlated with certain disease symptoms. The anti-OX40 antibodies described herein can carry detectable or functional tags. When using fluorescent tags, it is known in the art that currently available microscopy and fluorescence activated cell sorter analysis (FACS) or a combination of the two methods can be used to identify and quantify specific binding members. The anti-OX40 antibodies described here can carry fluorescent tags. Exemplary fluorescent tags include, for example, reactive and coupled probes (e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluorescent dyes (Alexa Fluor dyes, Cy dyes and DyLight dyes). Anti-OX40 antibodies can carry radioactive labels, such as the positions ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac, and ¹⁸⁶ Re. When using radioactive markers, currently available counting programs known in the art can be used to identify and quantify the specific binding of anti-OX40 antibodies to OX40 (e.g., human OX40). In the case of tag-based enzymes, detection can be accomplished by any of the currently used colorimetric, spectrophotometric, fluorescent spectrophotometric, amperometric titration or gas quantification techniques known in the art. This can be achieved by contacting the sample or control sample with an anti-OX40 antibody under conditions that take into account the formation of a complex between the antibody and OX40. Any complex formed between the antibody and OX40 is detected and compared in the sample and the control. In view of the specific binding of the antibodies described herein to OX40, the antibodies can be used to specifically detect the expression of OX40 on the cell surface. The antibodies described here can also be used to purify OX40 via immunoaffinity purification.

Also included here is an assay system, which can be prepared in the form of a test kit for quantitative analysis of, for example, the degree of presence of OX40 or OX40/OX40L complex. The system or test kit may include a labeling component (e.g., a labeling antibody) and one or more additional immunochemical reagents. See, for example, the following section 5.6 for a detailed description of the set.

In some aspects, provided herein is a method for detecting OX40 in a sample in vitro, the method comprising contacting the sample with an antibody. In some aspects, provided herein is the use of the antibodies provided herein to detect OX40 in a sample in vitro. In one aspect, provided herein is the antibody or pharmaceutical composition provided herein for use in detecting OX40 in a subject. In one aspect, there is provided the antibody or pharmaceutical composition provided herein for use as a diagnostic agent. In a preferred embodiment, the antibody includes a detectable label. In a preferred embodiment, OX40 is human OX40. In a preferred embodiment, the subject is a human.

5.6 Set

Provided herein are kits that include one or more of the antibodies or conjugates thereof described herein. In a specific embodiment, a drug pack or kit is provided here, which includes a drug pack filled with the medical composition described herein One or more containers of one or more ingredients (such as one or more antibodies provided herein). In some embodiments, the kit includes the pharmaceutical composition described herein and any prophylactic or therapeutic agents (such as those described herein). In certain embodiments, the kit may include T cell mitogens (such as phytohemagglutinin (PHA) and/or phorbol ethyl ester (PMA)), or TCR complex stimulating antibodies (such as anti-CD3 antibodies and anti- CD28 antibody). As the case may be, associated with such a container may be a label in a form regulated by a government agency that regulates the production, use, or sale of drugs or biological products, and the label reflects the approval of the production, use, or sale agency for human administration.

Also provided here are sets that can be used in the above methods. In one embodiment, the kit includes the antibodies described herein in one or more containers, preferably purified antibodies. In a specific embodiment, the kit described herein contains a substantially isolated OX40 antigen (e.g., human OX40), which can be used as a control. In another specific embodiment, the kit described herein further includes a control antibody that does not react with the OX40 antigen. In another specific embodiment, the kit described herein includes one or more elements for detecting the binding of an antibody to the OX40 antigen (e.g., the antibody can be conjugated to a detectable substrate (such as a fluorescent compound, an enzyme substrate) , A radioactive compound or a luminescent compound), or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In a specific embodiment, the kit provided herein may include recombinantly produced or chemically synthesized OX40 antigen. The OX40 antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detection device of the above kit includes OX40 anti The original attached solid support. Such kits may also include unattached, reporter-labeled anti-human antibodies or anti-mouse/rat antibodies. In this embodiment, the binding of the antibody to the OX40 antigen can be detected by the binding of the reporter-labeled antibody. In addition, a kit or a multi-part kit is provided, which includes (a) the antibody or pharmaceutical composition of the present invention and (b) a checkpoint targeting agent, IDO inhibitor, and/or vaccine.

The following examples are provided by way of illustration and not by way of limitation.

6. Examples

The examples in this section (i.e., section 6) are provided by way of explanation rather than limitation.

6.1 Example 1: Generation of novel antibodies against human OX40

This example describes the production and characterization of antibodies that bind to human OX40. Specifically, this example describes the production of human antibodies that specifically bind to human OX40 and exhibit costimulatory effects on T cells.

6.1.1 Library generation

The generation of the Retrocyte Display ^{TM library is described here.} For the generation of library inserts, total RNA was extracted from FACS-sorted CD19-positive human B lymphocytes derived from two cord blood samples by phenol/chloroform. Using the RevertAid first-strand cDNA synthesis kit from Fermentas (catalog numbers K1621 and K1622), total RNA from each cord blood sample (1 μg) was used for first-strand cDNA synthesis. The antibody variable region was amplified from cDNA by PCR and cloned into the retroviral expression vector (pCMA). These constructs were then used to transduce pre-B cells to express antibodies on the surface ^{using Retroocyte Display™ technology.} The retroviral expression vector contains 5'and 3'LTR, an immunoglobulin constant region (IGHG1 or IGKC) including a membrane anchor portion (IGHG1), and a CD4 surface marker gene.

Using a mixture of Vκ family-specific forward primers and reverse primers, the light chain variable region (VL) was amplified by semi-nested PCR. The forward primer introduced the Hind III selection site, and the reverse primer introduced the Eco 47III selection site.

Using a mixture of VH family-specific forward primers and reverse primers, the heavy chain variable region (VH) was amplified by PCR. The forward primer introduced the Hind III selection site, and the reverse primer introduced the Eco 47III selection site.

The amplified VH and Vκ regions were digested overnight at 37°C. After digestion, a band with a size of 400-450 bp was obtained and subjected to gel purification (Macherey & Nagel, NucleoSpin gel and PCR clean-up).

For the selection of the heavy chain variable region, construct 3181 (pCMA-InsX Cg(iso3)loxP2-I-tr_huCD4-loxP) was digested with Hind III/ Eco 47III at 37°C for 4 hours, and the size of 8362bp Strip gel purification. For the selection of the variable region of the kappa light chain, construct 3204 (pCMA-InsX Ck-I-CD4) was digested with Hind III/ Eco 47III at 37°C for 4 hours, and a band with a size of 7465 bp was gel purified .

Using a 1:3 ratio of vector to insert, the digested and purified antibody variable region is linked in frame to a suitable expression vector. Each VH and Vκ family was ligated into a retroviral expression vector and concentrated by precipitation 10-fold. The precipitated VH and Vκ ligation reactions were also transformed into E. coli DH10B cells for library production. The respective connection, precipitation, and transformation of each VH and Vκ family allows the library to reflect the natural distribution of functional germline genes, thereby ensuring that VH or VH genes with a large number of functional germline genes are compared to families with fewer functional germline genes. The Vκ family is highly representative in the final library. After transformation, E. coli cells were harvested and merged into the final library. Control the quality of each library by diagnostic restriction digestion and analysis of sequencing data. Calculate the library diversity from the sequence analysis data.

6.1.2 Recover heavy and light chains from pre-selected pre-B cell clones

The library materials generated as described above were used to identify antibodies with high binding affinity to OX40. The B cell clones were lysed, and the heavy and light chain variable regions were amplified from the inserted retroviral vector stably integrated into the genomic DNA using PCR methods standard in the art. The amplified heavy and light chain variable regions are then selected and colonized into mammalian expression vectors containing human heavy and light chain constant regions. The DNA plasmid preparation was then used to transfect CHO cells, and the expressed antibodies were tested using suspension array technology. In Microsynth (Balgach (Balgach), Switzerland (±) on the anti-heavy chain and light chain for sequencing.

6.1.3 Biophysical characterization of anti-OX40 antibodies

The antibody designated pab1949 was selected and characterized in multiple assays as described below. The anti-OX40 antibody pab1949 includes a heavy chain and a light chain, the heavy chain including the amino acid sequence of SEQ ID NO: 60 and the light chain including the amino acid sequence of SEQ ID NO: 50. _{The antibody pab1949 is a human IgG 1} antibody (according to Kabat) that contains a T109S substitution in the constant domain of the light chain (that is, the substitution of threonine to serine at position 109 relative to the constant domain of the wild-type light chain). Number), which helps to colonize the variable region in the frame into the constant region. The mutation system does not affect the binding or conservative modification of the antibody function. A wild-type counterpart containing threonine at position 109 (designated pab1949-1) (numbering according to Kabat) was also generated. _{The antibody pab1949-1 is a human IgG 1} antibody comprising the heavy chain of SEQ ID NO: 60 and the light chain of SEQ ID NO: 20.

6.1.3.1 Affinity determination by biofilm layer interference method

The affinity of pab1949-1 was determined by biofilm interferometry (BLI). In short, recombinant human OX40 antigen (OX40-Fc, R&D) was diluted with 1xPBS to obtain 1,000 μl of 0.2 μM and added to a 96-well plate. Dilute pab1949-1 in 1xPBS to a concentration of 50nM. A six-point serial dilution of pab1949-1 was prepared from a 50nM solution using 1xPBS to obtain antibody dilutions in the range of 50nM to 0.78nM, and the corresponding antibody serial dilutions were added to each well of a 96-well plate. According to the manufacturer's instructions, using the Octet ^® 16-channel mode, the sensor at 25 deg.] C was continued 5min coated with human OX40 antigen, the threshold value is 1.0nm. For blocking, incubate 0.5 mg/ml of non-specific IgG ₁ antibody for 10 minutes. Place the plate containing serial antibody dilutions of pab1949-1 on the Octet ^® instrument. Measure according to the manufacturer's instructions. Record the binding and dissociation of pab1949-1 and OX40 antigen for 3 minutes and 10 minutes, respectively. The data was analyzed using Octet ^® data analysis software, and the results are shown in Table 5.

6.1.3.2 Antibodies that bind to activated human or cynomolgus T cells

The binding properties of anti-OX40 antibodies pab1949 and pab1949-1 to human or cynomolgus OX40 were analyzed by flow cytometry.

Using magnetic-based separation (Miltenyi Biotec), enrichment of human PBMCs isolated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient was used unchanged CD4+ and CD8+ T cells. Then, under the recommended culture conditions, the enriched T lymphocytes were activated with CD3-CD28 expanded beads (Miltenyi Biotec) with 500 U rIL-2 (R&D Systems) for 3 days, and then 50 U rIL-2. The recommended culture conditions are defined as follows: _{cells are cultured in RPMI-1640 medium at 37°C and 5% CO 2} supplemented with 10% fetal bovine serum, 10 mM HEPES, and 1X Pen/Strep-glutamine. After activation, at 4°C, the cells were conjugated with antibodies containing CD3 (BV711, OKT3), CD4 (BV605, OKT4), CD8a (BV650, RPA-T8) and in FACS buffer (with 2% FBS). The surface antibody mixture of pre-conjugated anti-OX40 antibody or isotype control (both Afluor488, 10 μg/ml) diluted in PBS) was incubated for 30 minutes. Set aside additional samples for single dye compensation control (CD45-BV650, CD45-Afluor488, CD45-BV605, and CD45-BV711). Then, the cells were washed twice with FACS buffer and analyzed using the LSR Fortessa flow cytometer (BD Biosciences). Use the combination of FACS DIVA and WEHI Weasel software to analyze the flow cytometry chart. The anti-OX40 antibody pab1949 binds to activated human CD4+ T cells and CD8+ T cells (Figure 1A).

For binding activated T cells, a series of concentrations of pab1949-1 were tested to characterize the dose-response relationship. Briefly, human peripheral blood mononuclear cells (PBMC) were thawed and washed with PBS. Use magnetic beads (Miltenyi Biotec) for negative separation of T cells, and resuspend the purified T cells in RPMI+10% FBS and stimulate with anti-CD3/anti-CD28 beads _{at 37°C and 5% CO 2} Lasts 72 hours. The cells were washed and blocked with Fc blocking solution (Trustain, Biolegend) at room temperature for 15 minutes. The cells were washed again and stained with serial dilutions of pab1949-1 (10 to 0.00003 μg/ml) in the dark at 4°C for 45 minutes. The cells were washed and then combined with lineage marker antibodies including anti-CD3 fluorescein isothiocyanate (FITC) (clone SP34) and anti-CD4 Brilliant Violet (BV) 510 (clone OKT4). The two antibodies were stained together to detect pab1949-1 (anti-κIgG PE). The cells were washed, fixed with 1.6% paraformaldehyde, and collected using a Becton Dickinson Fortessa flow cytometer. pab1949-1 restricted binding to stimulated T cells only, and not to unstimulated T cells (Figure 1B). The binding of pab1949-1 to activated human CD4+ T cells was dose-dependent (Figure 1C).

Then, for the binding of pab1949-1, multiple quiescent immune cell subtypes were tested. The human PBMC were thawed and washed with PBS. To stain dead cells, add infrared (IR) viability dye and incubate at room temperature in the dark for 15 minutes. The cells were washed and stained with amine dye infrared (Life Technologies) for 15 minutes at room temperature. The cells were washed and subjected to Fc blocking (Trustain FcX, Biolegend) at room temperature for 10 minutes. After washing, at 4 ℃, the cells were treated with 1μg / ml of pab1949-1 or IgG ₁ isotype control incubated in the dark for 30 minutes. The cells were washed and stained with secondary reagents (anti-Fc F(ab')PE, Jackson Immune Research Laboratories), followed by lineage marker antibody staining, including anti-CD3 algae Erythrocyanine 7 (PECy7, clone SP34.2), anti-CD8 BV510 (clone SK1), anti-CD4 polymycin-chlorophyll-protein complex (PerCP) Cy5 (clone Ly200), and anti-CD8 CD14 FITC (clone TUK4). The cells were washed, fixed with 1.6% paraformaldehyde, and collected using a Becton Dickinson Fortessa flow cytometer. As shown in Figure ID, the anti-OX40 antibody pab1949-1 did not show detectable binding to CD14+ cells, CD4+ T cells, CD8+ T cells, CD20+ B cells, or CD3-CD20- cells.

To test species cross-reactivity, activated cynomolgus monkey ( Macaca fascicularis ) PBMC was used for cell binding assays. Briefly, at 37 ℃, atrioventricular humidified 5% CO _2, the viable cynomolgus monkey PBMC (Worldwide Primates Inc.) with Concanavalin -a (Sigma - Aldrich (Sigma Aldrich), 5μg/ml) and recombinant IL-2 (Miltenyi, 20U/ml) were activated in RPMI medium supplemented with 10% heat-inactivated FBS for 3 days. After activation, the cells were incubated with a human Fc receptor blocker (Biolegend) at room temperature for 15 minutes to reduce non-specific binding. The anti-OX40 antibody pab1949 or human IgG ₁ isotype control (10 μg/ml) was added to the sample and incubated at 4°C for 30 minutes. After washing once with FACS buffer, the APC-conjugated anti-human kappa antibody will be included together with antibodies specific for CD4 (BV605, OKT4) and CD8a (PE, RPA-T8) (both 2.5 μg/ml) The antibody mixture was diluted in FACS buffer (PBS, 2mM EDTA, 0.5% BSA and pH 7.2), added to each sample and incubated at 4°C for 30 minutes. Before dyeing, set aside additional samples for single dye compensation control (cyno-reactive: CD4-BV605, CD4-PE, and CD4-APC). The samples were washed twice in FACS buffer and analyzed using the LSR Fortessa flow cytometer (BD Biosciences). As shown in Figure 1E, pab1949 binds to activated cynomolgus CD4+ T cells.

6.1.3.3 OX40 antibody selective determination

The suspension array technology was used as a multiplex assay to evaluate the selectivity of pab1949-1 for OX40 relative to other members of the TNFR superfamily. Using standard NHS-ester chemistry, multiple TNFR family members are chemically linked to Luminex ^® microspheres. The purified material of pab1949-1 was diluted to 10ng/ml, 100ng/ml and 1000ng/ml in the assay buffer (Roche 11112589001). In short, each 25μl dilution was incubated in the dark (20°C, 650rpm) with 1500 Luminex ^® microspheres in 5μl assay buffer in a 96 half-well filter plate (Millipore, MABVN1250) 1 hour. Luminex ^® microspheres (Luminex Corp, LC10001-01, LC10005-01, LC10010-01, LC10014-01, LC10015-01, LC10018-01, LC10022-01, LC10026-01, LC10052- 01, LC10053-01 and LC10055-01) and recombinant human LTBR-Fc (Acros Biosystems, LTR-H5251), anti-human IgG (F(ab) ₂ -specificity, JIR, 105-006-097), recombinant Human OX40-Fc (R&D systems, 3388-OX), recombinant human GITR-Fc (R&D, 689-GR), recombinant human DR6-Fc (SinoBiological, 10175-H02H), recombinant human DR3-Fc (R&D, 943-D3), recombinant human GITR-His (SinoBiological, 13643-H08H), recombinant human TWEAK R-Fc (SinoBiological, 10431-H01H), recombinant human OX40-His (SinoBiological, 10481) -H08H), recombinant human 4-1BB-His (SinoBiological, 10041-H08H) or recombinant human BAFFR-Fc (R&D, 1162-BR) is coupled via an amine coupled to the surface of COOH beads. A 1:3 dilution series (0.08-540ng/ml) of 25 μl of human IgG1 standard (Sigma, 15154) in duplicate was used to generate a standard curve. Use 60μl goat anti-human IgGF(ab) ₂ labeled with R-PE (2.5μg/ml; JIR 109-116-098, AbDSerotec Rapid RPE antibody conjugate kit, LNK022RPE) and another hour of incubation time (20°C) , 650rpm) for detection. The plates were analyzed using the Luminex ^® 200 system (Millipore). In a sample volume of 48 μl, the total number of counts per well is 100 beads. The PE MFI value is used to determine the specific or non-specific binding to the aforementioned recombinant protein.

Antibody pab1949-1 showed specific binding to human OX40, and no significant binding to other TNFR family members was observed at the tested concentration (data not shown).

6.2 Example 2: Functional characterization of anti-OX40 antibodies

This example demonstrates the ability of the anti-OX40 antibodies pab1949 and pab1949-1 produced by the above method to act as agonists of OX40. The antibodies pab1949 and pab1949-1 were tested to determine their ability to co-stimulate primary human CD4+ or CD8+ T cells. In addition, _{pab1949 and pab1949-1, which are human IgG 1} antibodies, were converted into human IgG ₄ antibodies pab2044 and pab2044-1, respectively. Antibodies pab2044 and pab1949 have the same heavy chain variable region and the same light chain, but include the human IgG ₄ constant region. The antibody pab2044 includes the heavy chain sequence of SEQ ID NO: 61 and the light chain sequence of SEQ ID NO: 50. Similar to pab1949, pab2044 contains a single amino acid substitution of T109S in the constant region of the light chain and conservative modifications that do not affect antibody binding or function to promote colonization. The wild-type counterpart, pab2044-1, contains threonine at position 109 (numbering according to Kabat) and includes the heavy chain sequence of SEQ ID NO: 61 and the light chain sequence of SEQ ID NO: 20. Similarly, pab1949 and pab1949-1 were also converted into human IgG ₂ antibodies pab2193 and pab2193-1, respectively. The antibody pab2193 includes the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 50. The antibody pab2193-1 includes the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 20. In some assays, the functional activity of pab1949, pab1949-1, pab2044, pab2044-1, pab2193, or pab2193-1 was tested.

In some assays, the agonistic activity of the anti-OX40 antibody of the present invention was compared with the agonistic activity of reference antibodies pab1784 and pab2045. Antibody pab1784 was generated based on the variable region of antibody 11D4 provided in U.S. Patent No. 7,960,515 (herein incorporated by reference). The heavy chain of pab1784 includes the amino acid sequence of the heavy chain variable region of 11D4 (SEQ ID NO: 26) and the human IgG ₁ constant region of SEQ ID NO: 65. The light chain of pab1784 includes the amino acid sequence of the light chain variable region of 11D4 (SEQ ID NO: 24) and the constant region of SEQ ID NO: 25.

Antibody pab2045 was generated based on the variable region of antibody 20E5 provided in International Publication No. WO 13/038191 (incorporated herein by reference). The heavy chain of pab2045 includes the amino acid sequence of the heavy chain variable region of 20E5 (SEQ ID NO: 30) and the human IgG ₁ constant region of SEQ ID NO: 65. The light chain of pab2045 includes the amino acid sequence of the light chain variable region of 20E5 (SEQ ID NO: 28) and the constant region of SEQ ID NO: 41.

6.2.1 The effect of anti-OX40 antibody against CD3 stimulated CD4+ T cell proliferation

In order to test the effect of pab1949 on T cell proliferation, magnetic-based separation (Stemcell Technologies) was used, and the unchanged CD4+ T cells were removed from the buffy coat of healthy donors via a ficoll gradient (Research Human PBMC isolated in Blood Components), LLC) are enriched. Cell proliferation is determined by monitoring the dilution of carboxyfluorescein acetyl succinimidyl acetate (CFSE) dye in dividing cells (Quah BJ et al., (2007) Nat Protoc, 2( 9): 2049-56). At 37°C, the enriched CD4+ T cells were ^{labeled with 10 μM CellTrace™} CFSE (Life Technologies) for 7 minutes. After thorough washing, the cells were suspended in RPMI1640 medium supplemented with 10% heat-inactivated FBS at 1 x 10 ⁶ cells/ml. The total of 100μl (1 x 10 ⁵ cells) were inoculated with the anti-CD3 antibody (3μg / ml, BD Biosciences (BD Biosciences)) with 5μg / ml of pab1949,5μg / ml of the IgG1 isotype control, or 2μg /ml of anti-CD28 antibody (BD Biosciences) pre-coated flat bottom 96-well plate in each well, and cultured _{at 37°C and 5% CO 2.} On the 5th day, the cells were stained with 0.5μl/well of anti-CD4 antibody labeled with PerCP-Cy5.5 in FACS buffer (2% FBS in PBS) for 30 minutes at 4°C, and by Flow cytometry on LSRFortessa (BD Biosciences) to determine the percentage of CFSE low CD4+ cells. Use FlowJo to analyze flow cytometry data.

Use the same as described above in which CD4+ T cells labeled with CFSE are inoculated with anti-CD3 antibody (3μg/ml, BD Biosciences) and 5μg/ml pab2044, 5μg/ml IgG ₄ Type control (pab2031), or 2μg/ml anti-CD28 antibody (BD Biosciences) pre-coated 96-well plate in a similar assay to evaluate the activity of pab2044. On day 5, the percentage of CFSE low CD4+ cells was checked by flow cytometry.

Figures 2A and 2B are bar graphs derived from a representative flow cytometric analysis of CD4+ T cell proliferation induced by co-stimulation with anti-OX40 antibody, showing the cell number (Y axis) and CD4+ T cells labeled with CFSE The level of fluorescence emitted (X axis). With increased fineness The percentage of cells is accompanied by a decrease in the level of fluorescence emitted by CFSE, showing enhanced CD4+ T cell proliferation. The bar graph indicates the percentage of CFSE low CD4+ cells. When added to cells activated with suboptimal concentrations of anti-CD3 antibodies, pab1949 (Figure 2A) and pab2044 (Figure 2B), when bound to the plate, both induced CD4+ T cell proliferation.

Then, the dose response of pab1949 in inducing T cell proliferation was measured. Using magnetic-based separation (Stemcell Technologies), for unchanged CD4+ T cells, PBMCs separated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient Enriched. Then, the enriched clusters of CD4+ T cells were ^{labeled with 10 μM CellTrace TM} CFSE (Life Technologies) at 37° C. for 7 min. After thorough washing, the cells were suspended in RPMI1640 medium supplemented with 10% heat-inactivated FBS at 1 x 10 ⁶ /ml. Inoculate 100 μl (1 x 10 ⁵ ) of cells into a flat-bottom 96-well plate pre-coated with anti-CD3 antibody (3 μg/ml, BD Biosciences) and different concentrations of pab1949 or IgG _{1 isotype control} In each well, and incubate at 37°C and 5% CO ₂ . On the 4th day, the cells were stained with 0.5μl/well of APC-labeled anti-CD4 antibody in FACS buffer (2% FBS in PBS) for 30 minutes at 4°C, and treated with LSRFortessa (BD Biotech Company (BD Biosciences)) to determine the percentage of CFSE low CD4+ cells.

As shown in Figure 2C, the anti-OX40 antibody pab1949 can be Maintain high levels of T cell proliferation at pharmacologically relevant antibody concentrations. CD4+ T cell proliferation is a substantially increasing function of pab1949 concentration between 0.2 μg/ml and 20 μg/ml (Figure 2C).

6.2.2 The effect of anti-OX40 antibody on the production of human PBMC cytokines stimulated by CD3

As further evidence of the agonistic activity of the anti-OX40 antibodies pab1949 and pab1949-1, the production of cytokines under suboptimal anti-CD3 stimulation was measured.

For intracellular cytokine staining experiments, human PBMCs isolated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient were stored in liquid nitrogen, and Thaw on the day of the experiment. Resuspend the cells in cell culture medium (RPMI+10% FBS+20U/mL for IL-2) and add them to a 96-well culture plate containing different suboptimal concentrations of anti-CD3 antibodies bound to the plate , And 5μg/ml anti-OX40 antibody pab1949 or isotype control IgG ₁ antibody. The samples were incubated at 37°C and 5% CO _{2 for 3 days.} After activation, in order to limit protein transport within the cells, the cells were treated with Brefeldin A (BD Biosciences) according to the manufacturer’s instructions and kept at 37°C and 5% CO ₂ , Incubate the sample for 6 hours. After culturing, the dead cells were stained with viability amine dye (Life Technologies). After washing with FACS buffer (PBS, 2% FBS, pH 7.2), the inclusions diluted in cold FACS buffer are specific for CD3 (APC Cy7, SP34.2), CD4 (PercP Cy5.5, L200) The antibody mixture of, and CD8a (PE Cy7, SK1) antibodies was added to each sample and incubated at 4°C for 10 minutes. The cells were fixed and permeabilized with Cytofix-Cytoperm (BD Biosciences) for intracellular staining according to the manufacturer's instructions. PBMC were stained with antibodies specific for IFNγ (Alexa647, B27) and TNFα (PE, Mab11), and incubated at room temperature for 10 minutes. Before staining, using a single-stain compensation control, the beads bound to the mouse IgG antibody kappa light chain were stained with the antibody used to stain the cells. The samples were washed with 1xPerm-wash buffer (BD Biosciences) and analyzed using a FACS Canto flow cytometer (BD Biosciences). Use Flojo software to analyze flow cytometry graphs.

PBMC from four different donors were tested: donor KM, donor TM, donor GS, and donor SB. For all donors, pab1949 demonstrated costimulatory activity on human T cells, including IFNγ+ TNFα+ multifunctional CD4+ T cells and CD8+ T cells, as well as TNFα+ monofunctional CD4+ T cells and CD8+ T cells (Figure 3A, 3B, and 3C). In the PBMC from the donor GS, pab1949 can also increase the percentage of IFNγ+ monofunctional T cells (Figure 3B).

Then, in a suboptimal anti-CD3 stimulation assay similar to the above-mentioned assay using PBMC derived from donor GS, the dose titration of the anti-OX40 antibody pab1949-1 was tested. In short, at 37°C and 5% CO ₂ , PBMC and anti-CD3 antibody bound to the plate _{(0.8 μg/ml) and pab1949-1 or IgG 1} isotype control antibody bound to the plate (0, 0.3, 1, 3, 6, 12, 25, or 50μg/ml) for 4 days. After activation, in order to limit protein transport within the cells, the cells were treated with Brefeldin A (BD Biosciences) according to the manufacturer’s instructions and kept at 37°C and 5% CO ₂ , Incubate the sample for 6 hours. After culturing, the cells were stained with FITC viability amine dye (Life Technologies) to distinguish live cells from dead cells. After washing with cold buffer (1xPBS+2% FBS, pH 7.2), it will contain anti-CD3 (APC Cy7, SP34.2), anti-CD4 (PercP Cy5.5, L200), and anti-CD8a (PE Cy7, SK1) The antibody mixture of) was added to each sample and incubated at 4°C for 10 minutes. The cells were fixed and permeabilized with Cytofix-Cytoperm (BD Biosciences) for intracellular staining according to the manufacturer's instructions. PBMCs were stained with anti-IFNγ (Alexa647, B27) and anti-TNFα (PE, Mab11) antibodies, and incubated at room temperature for 10 minutes. The samples were washed with 1xPerm-wash buffer (BD Biosciences) and collected using a FACScanto flow cytometer (BD Biosciences). Use Flojo software to analyze flow cytometry graphs. As shown in Figure 3D-3F, anti-OX40 antibody pab1949-1 demonstrated costimulatory activity and increased TNFα+ CD4+ T cells, IFNγ+ TNFα+ multifunctional CD8+ T cells, and IFNγ+ CD8+ T cells in a dose-dependent manner. percentage.

The costimulatory activity of the pab1949-1 dose range was further tested using cells derived from PBMCs of additional donors in the suboptimal anti-CD3 stimulation assay described above. _{The anti-OX40 antibody pab1949-1 and the IgG 1} isotype control antibody were tested at 0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml. The anti-OX40 antibody pab1949-1 consistently increased the percentage of IFNγ+ and/or TNFα+ T cells in PBMC from multiple donors (Figure 4A-4C).

It is worth noting that for PBMCs from many donors, the percentage of IFNγ+ and/or TNFα+ T cells induced by the anti-OX40 antibody pab1949-1 is a substantially incremental increase in the antibody concentration within the wide range of antibody concentrations tested. Functions (Figures 3D-3F and 4A-4C).

To further test the agonistic activity of the anti-OX40 antibody pab1949, the amount of secreted cytokines was measured. Human PBMC isolated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient were stored in liquid nitrogen and thawed on the day of the experiment. Resuspend the cells in cell culture medium (RPMI+10% FBS+20U/mL for IL-2) and add them to a 96-well culture plate containing different suboptimal concentrations of anti-CD3 antibodies bound to the plate , And 5μg/ml anti-OX40 antibody pab1949 or isotype control IgG ₁ antibody. The samples were incubated at 37°C and 5% CO ₂ and the cell culture supernatant was collected after 4 days (SB#1A) or 3 days (SB#1B, SB#2, and GS). According to the manufacturer's instructions, the samples were tested using the V-PLEX Proinflammatory Panel (human) kit (Meso Discovery Company) for the production of IL-2, TNFα, IL-10, IL-4, and IL-13.

As depicted in Figure 5A, the anti-OX40 antibody pab1949 costimulates the production of cytokines in human PBMC from two different donors (donor SB and donor GS). The cytokine production of PBMC from the donor SB was tested in two separate experiments: SB#1A and SB#1B show the results from the first experiment, which were measured after 4 and 3 days of stimulation, respectively Cytokines; and SB#2 shows the results from the second experiment in which cytokines are measured after 3 days of stimulation.

Then, using cells derived from PBMC of the donor GS in the suboptimal anti-CD3 stimulation assay similar to the above assay, the secretion of cytokines induced by the dose titration of pab1949-1 was tested. In short, under 37℃ and 5% CO ₂ , PBMC and anti-CD3 antibody bound to the plate (0.8μg/ml) and pab1949-1 or IgG ₁ isotype control antibody bound to the plate (0, 0.3, 1, 3, 6, 12, 25, or 50μg/ml) for 4 days. After activation, the cell culture supernatant was collected, and the human TH1/TH2 10-Plex tissue culture kit (Meso Discovery) was used to detect cytokines.

As shown in Figures 5B-5D, the anti-OX40 antibody pab1949-1 stimulated the production of TNFα, IL-10, and IL-13 in a dose-dependent manner.

Cells derived from PBMC from another donor were used to further confirm the costimulatory activity of pab1949-1 in inducing the secretion of cytokines. In short, under 37°C and 5% CO ₂ , PBMC and anti-CD3 antibody bound to the plate _{(0.8 μg/ml) and pab1949-1 or IgG 1} isotype control antibody bound to the plate (0, 0.7, 1.6, 3.1, 6.3, 12.5, 25, or 50μg/ml) for 4 days. After activation, the non-human primate (NHP) V-Plex assay kit (Meso Discovery) was used to measure the amount of cytokines secreted into the supernatant.

For all donors tested, pab1949-1 dose-dependently increased the secretion of GM-CSF (Figures 6A-6C), IL-2 (Figures 7A-7C), and TNFβ (Figures 8A-8C).

For PBMC from many donors, the cytokines (GM-CSF, IL-2, TNFα, TNFβ, IL-10, and IL-13) induced by the anti-OX40 antibody pab1949-1 secrete antibody concentrations in a wide range of antibodies A substantially increasing function within the concentration (Figures 5B-5D, 6A-6C, 7A-7C, and 8A-8C).

6.2.3 The role of anti-OX40 antibodies in effector T cells: regulatory T cell co-culture assays

Then, the activity of the anti-OX40 antibody pab1949-1 in the effector T cell (Teff): regulatory T cell (Treg) co-culture assay was tested. In short, human PBMCs isolated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient were stored in liquid nitrogen and thawed on the day of the experiment. Separate regulatory T cells and effector T cells by magnetic bead separation (respectively CD4 ⁺ CD25 ⁺ CD127 ^dim/- regulatory T cell separation kit II and Pant T cell kit, Miltenyi Biotec). Then, by culturing the anti-CD3/anti-CD28/anti-CD2 beads (Miltenyi Biotec) in the cell culture medium (RPMI+10% FBS) at a ratio of 1:2 (T cells: beads) T cell activation for 2 days. After activation, in anti-CD3/anti-CD28/anti-CD2 beads, soluble or cross-linked (using anti-Fc F(ab') ₂ , Jackson ImmunoResearch) pab1949-1 or IgG _{1 In} the presence of an isotype control (10μg/ml), add regulatory T cells and effector T cells to a 96-well culture plate at a ratio of 1:3 (Treg:Teff). The samples were incubated for 4 days at 37°C and 5% CO _2. Following activation, supernatants were collected and used AlphaLISA ^® (PerkinElmer, Inc. (Perkin Elmer)) or measuring IL-10 IL-2.

In this in vitro Teff:Treg co-culture assay, the anti-OX40 antibody pab1949-1 alleviated the inhibition of the Teff cell population by Treg cells, such as the enhancement in cells treated with pab1949-1 compared with cells treated with isotype As evidenced by IL-2 production (Figure 9A) and reduced IL-10 production (Figure 9B).

6.2.4 The effect of anti-OX40 antibody on human PBMC under staphylococcal enterotoxin A (SEA) stimulation

After staphylococcal enterotoxin A (SEA) stimulation, the functional activities of anti-OX40 antibodies pab1949 and pab1949-1 on primary human PBMC were further evaluated. Will cryogenic penicillin, streptomycin and 10% FBS (Hyclone) in RPMI1640 supplemented saved in PBMC (10 ⁵ cells / well) were added to 96-well NUNCLON delta planar plates (NUNC ^TM). At 37°C, 5% CO ₂ and 97% humidity, in the absence or presence of a fixed concentration (10μg/ml in Figures 10A and 10B) or different concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128) μg/ml in Figure 10C and 10D; 50, 10, 2, 0.4, 0.08, 0.016, and 0.0032 μg/ml in Figure 10E) anti-OX40 antibody or isotype control and 100ng/ml SEA (Toxin Technologies) In the case, culture the cells for 5 days. The clear supernatant was collected and stored at -80°C until analysis. For IL-2 and IL-10, electrochemiluminescence (Meso Discovery) is used to generate the titers of cytokines.

The anti-OX40 antibody pab1949 showed agonistic activity in this primary human PBMC assay, including IL-2 production (Figure 10A) and inhibition of IL-10 production (Figure 10B). The enhancement of IL-2 production by pab1949 at 10 μg/ml was superior to that observed with reference anti-OX40 antibodies pab1784 and pab2045 (Figure 10A). Figures 10C, 10D, and 10E are dose response curves from three independent experiments showing the fold change of IL-2 after co-stimulation with different concentrations of pab1949, pab1949-1, or reference antibodies pab1784 and pab2045. The antibodies pab1949 and pab1949-1 exhibited a different dose response relationship from the reference antibody and were able to induce high levels of IL-2 production at pharmacologically relevant antibody concentrations. IL-2 production induced by pab1949 or pab1949-1 is a substantially increasing function of antibody concentration in a wide range of antibody concentrations (for example, between 0.032 and 20 μg/ml, as shown in Figures 10C and 10D, or in Between 0.0032 and 50 μg/ml, as shown in Figure 10E).

Then, the functional activities of the _{IgG 1} antibody pab1949-1 and the IgG ₂ antibody pab2193-1 were compared in the above-mentioned primary human PBMC assay. Briefly, cryopreservation of human PBMC (Blood Research Department (Research Blood Components)) at 10 ¹⁰⁵ cells / well were plated in medium supplemented with Normocin ^TM (Invitrogen (Invivogen) in 96-well plates NUNCLON delta plane, # ant-nr) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) RPMI1640 medium. At 37°C, 5% CO ₂ and 97% humidity, the cells are the same species _{as pab1949-1, pab2193-1, and IgG 1} with increasing concentrations (50, 10, 2, 0.4, 0.08, 0.016, and 0.0032μg/ml) Type control antibody, or IgG ₂ isotype control antibody, and 100ng/mlSEA superantigen (Toxin Technologies) were cultured for 5 days. The clear supernatant was collected and stored at -80°C until analysis. The concentration of IL-2 was measured by ELISA.

As shown in Figure 10F, both the IgG ₁ antibody pab1949-1 and the IgG ₂ antibody pab2193-1 induced IL-2 production in human PBMC. Similar to pab1949-1, pab2193-1 also exhibits a dose-response relationship, where IL-2 production is a substantially increasing function of antibody concentration.

In addition, by comparing the IgG ₁ antibody pab1949-1 with the unglycosylated variant pab1949-1-N297A, the role of FcγR interaction in the functional activity of the anti-OX40 antibody pab1949-1 was examined. pab1949-1-N297A shares the heavy chain variable region and light chain sequence with pab1949-1, but includes the N297A substitution (numbering according to the EU numbering system) in the heavy chain constant region.

Human PBMC isolated from healthy donor buffy coat (Research Blood Components, LLC) via a ficoll gradient were stored in liquid nitrogen and thawed on the day of the experiment. Resuspend the cells in cell culture medium (RPMI+10% heat-inactivated FBS) and use 100ng/ml SEA (Toxin Technologies) and pab1949-1, pab1949-1-N297A, _{at 37°C and 5% CO 2} Or IgG ₁ isotype control antibody (0-50μg/ml) dose titration and culture for 5 days. The supernatant was collected, and then AlphaLISA ^® (PerkinElmer, Inc. (Perkin Elmer)) testing IL-2.

As shown in Figure 10G, pab1949-1 and pab1949-1-N297A both induced IL-2 production in human PBMC in a dose-dependent manner when stimulated by SEA. The key glycosylation of the single n-linked glycosylation site present at aspartame 297 (N297) was deleted on the pab1949-1-N297A variant antibody, which resulted in the lack of binding of its Fc fragment to FcγR. Compared to the wild-type counterpart, this variant antibody exhibits reduced agonistic activity.

6.2.5 Effect of agonistic anti-OX40 antibody on OX40 NF-κB-luciferase reporter cell line

The human OX40 NF-κB-luciferase reporter cell line was used to measure the ability of the anti-OX40 antibody pab1949-1 to mediate signal transduction in T cells. Resuspend the reporter cells generated using the Jurkat cell line in the assay medium (RPMI+10% FBS+penicillin/streptomycin/glutamate+1μg/ml puromycin), and use different concentrations of soluble pab1949-1 (0-6μg / ml) or isotype control IgG ₁ antibody in the presence of (complex condition) or cultured in the presence of (soluble conditions) anti-Fc reagent. Incubate the plate for 2 hours at 37°C and 5% CO _2. After incubation, the plate was equilibrated at room temperature, and then an equal volume of room temperature Nano-Glo reagent (Promega) was added. Use EnVision Multi-Mark Reader 2100 to read the luminescence.

Only the cross-linked pab1949-1 induced significant activation of the OX40 NF-κB-luciferase reporter cell line (Figure 11B). Soluble pab1949-1 induced the lowest activation of the reporter cell line, and the IgG ₁ isotype control antibody did not induce detectable levels of luciferase expression (Figures 11A and 11B).

6.2.6 The effect of agonistic anti-OX40 antibody on Fcγ receptor IIIA reporter cell line

In this example, a reporter cell line expressing Fcγ receptor IIIA and a target cell expressing human OX40 are used to co-engage OX40 and communicate with _{IgG 1} antibody pab1949-1 and IgG _{4 antibody pab2044-1 via activated Fcγ receptors.} Evaluation. Jurkat NFAT-luciferase reporter cells (Promega) overexpressing FcγRIIIA (158 V/V polymorphism) were used as effector cells. The antibody/antigen complex, where the antigen is located on the surface of the target cell, binds to the FcγRIIIA on the effector cell to signal the promoter/reporter construct and result in luciferase gene expression.

The dose of OX40-overexpressing cells (PHA-activated Hut102 cells) and FcγRIIIA reporter cells in soluble pab1949-1, pab2044-1, IgG ₁ isotype control, or IgG ₂ isotype control (0-10 μg/ml) Co-cultivation in the presence of titration. According to the manufacturer's instructions, the reporter cell activation is evaluated and the relative light unit (RLU) is recorded. The △RLU is calculated as follows: the RLU of the anti-OX40 antibody minus the RLU of the isotype control. , When bound to cells expressing OX40-, only the IgG ₁ antibody pab1949-1 reporter cell activation FcγRIIIA FIG 12A.

6.2.7 Effect of agonistic anti-OX40 antibody on Fcγ receptor IIA reporter cell line

Then, use reporter cell line expressing FcγRIIA (Promega) and target cells (Jurkat cells expressing human OX40) to IgG ₁ antibodies pab1949-1 and pab1949-1-S267E/L328F together with IgG ₂ antibodies The ability of pab2193-1 to co-engage OX40 and communicate via FcγRIIA was evaluated. pab1949-1-S267E/L328F shares heavy and light chain sequences with pab1949-1, but includes S267E and L328F substitutions in the constant region of the heavy chain (numbering according to the EU numbering system).

Jurkat cells expressing FcγRIIA with high affinity 131 H/H polymorphism and NFAT response elements driving the expression of firefly luciferase were used as effector cells. In short, 25 μl of target cells (6 x 10 ⁶ cells/ml) and 25 μl of serially diluted antibodies were mixed in the double well of a 96-well white assay plate. The tested anti-systems pab1949-1, pab1949-1-S267E/L328F, pab2193-1, IgG ₁ isotype control antibody and IgG ₂ isotype control antibody. Then, 25 μl of effector cells (6 x 10 ⁶ cells/ml) were added to each well to obtain an effector: target ratio of 1:1. Incubate the plate for 20 hours at 37°C and 5% CO _2. After this incubation, Bio-Glo Luciferase Assay Reagent (Promega) was thawed at room temperature, and 75 μl was added to each well. Within 5-10 minutes, the luminescence was measured using an EnVision multi-label microplate detector (PerkinElmer). The background luminescence is subtracted from each sample reading, and the adjusted relative light unit (RLU) is recorded.

As shown in Figure 12B, the IgG ₂ antibody pab2193-1 showed the strongest FcγRIIA ^H131 activation when bound to cells expressing OX40, followed by pab1949-1-S267E/L328F and pab1949-1.

6.2.8 The interaction of anti-OX40 antibodies with regulatory T cells or effector T cells

In this example, the expression of human OX40 by activated natural regulatory T cells (nTreg) and effector T cells (Teff) was tested. Using magnetic-based separation, PBMCs isolated from healthy donors are enriched for unchanged CD3+ T cells (Teff) or CD4+ CD25+ CD45RA+ T cells (nTregs). T lymphocytes were activated with anti-CD3/CD28-conjugated beads with 500 U rIL-2 for 4 days, and reactivated with 50 U rIL-2 for 4 days. After 8 days of activation, T cells were harvested and stained with dead cells/live cells fixable Near-IR dead cells in PBS for 20 minutes at 4°C. It will include CD4 (BV605, OKT4), CD8a (BV650, RPA-T8), CD127 (BV421, AO1905), CD25 (APC, M-A251), and OX-40 (PE, ACT35), diluted in a buffer (PBS with 2% FBS) surface antibody mixture of antibody conjugated to each The samples were incubated at 4°C for 30 minutes. Then, the cells were washed and fixed with a buffer and stained with an intracellular antibody mixture containing conjugated antibodies against CD3 (BV711, OKT3) and Foxp3 (AF488, PCH101) diluted in a buffer. For OX40, a mouse anti-human IgG1-PE isotype control was used, and a sample from each T cell population was also stained with a fluorescence minus one (FMO) control. The samples are analyzed by flow cytometry. According to the manufacturer's instructions, PE-conjugated Quantibrite beads were run at the same time and used to quantify the OX40 receptor density.

As shown in Figure 13A, the surface performance of human OX40 on activated nTreg cells is higher than its surface performance on activated CD4+ or CD8+ effector T cells.

In a similar study, activated nTreg and Teff from two donors were stained with commercial anti-OX40 antibody (BER-ACT35 clone) or isotype control antibody and analyzed by flow cytometry. △Mean fluorescence intensity (△MFI) represents the MFI of the anti-OX40 antibody minus the MFI of the isotype control. The results are shown in Figure 13B.

Then, using the aforementioned reporter cell line expressing Fcγ receptor IIIA (FcγRIIIA) and the activated effector T cells (Teff) or nTreg cells generated as above, the anti-OX40 antibody pab1949 was performed through the ability of activated Fcγ receptors to co-engage OX40 and communicate. Evaluation. The anti-OX40 antibody pab1949 or IgG ₁ isotype control was serially diluted with a 3-fold dilution with a total concentration of 10 μg/ml. In a double well, 25 μl of each antibody dilution was added to Teff or nTreg cells. Jurkat NFAT-luciferase reporter cells overexpressing FcγRIIIA (158 V/V polymorphism) were added at an effector-target ratio of 1:1. The plate was incubated for 20 hours, and then analyzed using Bio-Glo Luciferase Assay Reagent (Promega). The background luminescence (blank outer hole) is subtracted from each sample reading, and the adjusted relative light unit (RLU) is recorded. ΔRLU is shown in Figure 13C, which represents the RLU of the anti-OX40 antibody minus the RLU of the isotype control.

Using a lightly modified experimental protocol, the study depicted in Figure 13C was repeated. In short, the buffy coat (Research Blood Components) from healthy volunteers was used to isolate primary regulatory T cells and effector T cells. Two T cell subsets were purified by magnetic bead separation (respectively CD4 ⁺ CD25 ⁺ CD127 ^dim/- regulatory T cell isolation kit II and Pant T cell kit, Miltenyi Biotec), and then by combining the cells with Anti-CD3/anti-CD28/anti-CD2 beads (Miltenyi Biotech) were cultured in a cell culture medium (RPMI+10% FBS) at a ratio of 1:4 (T cells: beads) for activation for 7 days. The activated Treg cells or Teff cells and the above-mentioned Jurkat NFAT-luciferase reporter cells (Promega) expressing FcγRIIIA were combined with soluble pab1949-1 or IgG ₁ isotype control antibody (0-10μg/ ml) co-cultivation in the presence of dose titration. The activation of reporter cells was evaluated according to the manufacturer's instructions, and ΔRLU is shown in Figure 13D.

Consistent with the difference between activated nTreg and activated CD4+ or CD8+ effector T cells, the surface OX40 performance is consistent (Figures 13A and 13B), the anti-OX40 antibodies pab1949 (Figure 13C) and pab1949-1 (Figure 13D) are preferably labeled Activated nTreg cells, thereby inducing FcyRIIIA-dependent signal transduction in the reporter cell line.

In order to evaluate whether OX40 overexpression is a characteristic of regulatory T cells located within the tumor microenvironment, comparisons were made in the blood isolated from healthy human donors (Figure 14A, ac, n=3) or isolated from non-small cell lung cancer (NSCLC) OX40 performance on T cells of the patient's tumor tissue (Figure 14A, df, n=3). In order to eliminate the background binding of antibodies to the immune population, all cells were incubated with purified CD16/32 antibody (10 μg/ml, 20 minutes at room temperature) before adding cell surface and intracellular antibodies. After FcR-blocking, all samples were used APC-conjugated anti-OX40 antibody (clone Ber-ACT35) or isotype control and cell surface antibody lineage mixture (CD3-FITC, CD25-PECy7, CD4-BV650 and CD8a- PE) incubate on ice for 45 minutes (each 1μg/ml), wash three times with FACS buffer (PBS, EDTA, and 0.5% BSA), then fix/permeabilize and use Pacific Blue-conjugated FOXP3 (fixed on ice /Permeabilization and culture for 45 minutes each, 1μg/ml) culture. Then, the stained samples were analyzed using the LSR Fortessa flow cytometer (BD Biosciences). The cell population in Figure 14A is defined as follows: Tconv (CD3+, CD4+, CD8a-, CD25 low, FOXP3-) or Treg (CD3+, CD4+, CD8a-, CD25 high, FOXP3+).

As shown in Figure 14A, the surface expression of OX40 is highest on regulatory T cells isolated from tumor tissues of NSCLC patients, while there is very low or no detectable level on Treg or conventional T cells from healthy donors.

For other tumor types, a similar analysis is performed. In short, the frozen and dissociated tumor samples (Conversant) or PBMC are thawed in AutoMACS washing solution (washing buffer, Miltenyi Biotec), and the cells are Fc-blocked (Trustain FcX, Biological Legend) before staining the cell surface. Company (Biolegend)). The cells were washed with washing buffer and stained with lineage marker antibodies at 4°C for 45 minutes. The lineage marker antibodies included anti-CD3 (clone SP34), anti-CD4 (clone OKT4), and anti-CD8 (Clone SK1), anti-CD25 (clone MA-251), and anti-OX40 (clone BER-ACT35). According to the manufacturer's instructions, the cells were washed and permeabilized with the forkhead box P3 (FOXP3)/transcription factor staining buffer set (eBioscience). After permeabilization, the cells were stained with anti-FOXP3 eFluor450 (clone PCH101, eBioscience). BD Biosciences (BD Biosciences) Fortessa flow cytometer was used to collect stained samples, and Flojo software was used to analyze the data.

Samples from multiple tumor types (including ovarian cancer, colon cancer (CRC), endometrial cancer, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), and breast cancer) The performance of OX40 in nodal T cells was higher than that in effector T cells associated with tumors (Figures 14B, 14C, and 14D).

6.2.9 The effect of anti-OX40 antibody on the production of cytokines in cynomolgus monkey PBMC stimulated by CD3

Then, using the sub-optimal anti-CD3 stimulation assay, the anti-OX40 antibody pab1949-1 was tested for the agonistic activity of cynomolgus monkey PBMC. In short, frozen cynomolgus PBMC (World Wide Primates) were stored in liquid nitrogen and thawed on the day of the experiment. Resuspend the cells in cell culture medium (IL-2 RPMI+10% FBS+20U/ml), and at 37°C and 5% CO ₂ , combine them with the plate-bound anti-CD3 antibody (0.8μg/ml ) And the pab1949-1 or IgG ₁ isotype control antibody (0, 0.8, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) bound to the plate for 4 days. The cell culture supernatant was collected, and the non-human primate (NHP) V-Plex assay kit (Meso Discovery Company) was used to test the secreted cytokines.

The anti-OX40 antibody pab1949-1 dose-dependently enhanced GM-CSF (Figures 15A and 15B), IL-17 (Figures 16A and 16B), TNFβ (Figures 17A and 17B), IL-5 (Figures 18A and 18B), And the production of IL-10 (Figures 19A and 19B) in the PBMC of multiple cynomolgus monkeys.

6.2.10 The effect of anti-OX40 antibody on PBMC of cynomolgus monkeys when staphylococcal enterotoxin A (SEA) is stimulated

After staphylococcal enterotoxin A (SEA) stimulation, the ability of pab1949-1 to co-stimulate PBMC of cynomolgus monkeys was further analyzed. The frozen cynomolgus monkey PBMC (World Wide Primates) were stored in liquid nitrogen and thawed on the day of the experiment. The cells were resuspended in cell culture medium (RPMI + 10% heat-inactivated FBS) at 37 [deg.] C and are and 5% CO _2, with SEA antigen (100ng / ml) together with pab1949-1 or IgG ₁ isotype control antibody ( 0, 0.8, 1.6, 3.1, 6.3, 12.5, 25, or 50 μg/ml) dose titration for culture for 5 days. After activation, the cell culture supernatant was collected, and the non-human primate (NHP) V-Plex assay kit (Meso Discovery) was used to test the secreted cytokines.

As shown in Figures 20A and 20B, the anti-OX40 antibody pab1949-1 increased IL-2 production in cynomolgus monkey PBMC from two donors.

6.3 Example 3: Epitope mapping of anti-OX40 antibody

In this example, the epitopes of pab1949 and the reference anti-OX40 antibody pab1928 were analyzed by alanine scan. The antibody pab1928 was generated based on the variable region of the antibody Hu106-122 provided in U.S. Patent Publication No. US 2013/0280275 (incorporated herein by reference). The heavy chain of pab1928 includes the amino acid sequence of the heavy chain variable region of Hu106-122 (SEQ ID NO: 56) and the human IgG1 constant region of SEQ ID NO: 65. The light chain of pab1928 includes the amino acid sequence of the light chain variable region of Hu106-122 (SEQ ID NO: 57) and the amino acid sequence of SEQ ID NO: 25 Constant region. Therefore, the heavy chain includes the amino acid sequence of SEQ ID NO:72 and the light chain includes the amino acid sequence of SEQ ID NO:59.

6.3.1 Epitope Mapping-Alanine Scan

The binding properties of pab1949-1 and the reference antibody pab1928 were determined by alanine scan. In short, the QuikChange HT Protein Engineering System (QuikChange HT Protein Engineering System) from Agilent Technologies (G5901A) was used to generate human OX40 mutants with alanine substitution in the extracellular domain. Using standard transfection techniques, followed by transduction as described above, human OX40 mutants were expressed on the surface of 1624-5 cells.

For cells exhibiting correctly folded human OX40 mutants, as demonstrated by the combination of multiple anti-OX40 antibodies in flow cytometry, further selections were made that exhibited human OX40 mutants, and did not bind to monoclonal anti-OX40 antibodies pab1949-1 or Subgroup of pab1928. By preparative, high-speed FACS (FACSAria II, BD Biosciences), cells exhibiting specific antibody binding are separated from the non-binding cell population. Expand the antibody-reactive or non-reactive cell pool again in tissue culture. Due to the stable phenotype of retrovirus-transduced cells, repeat the cycle of antibody-directed cell sorting and tissue culture amplification until a clearly detectable Anti-OX40 antibody (pab1949-1 or pab1928) at the point of non-reactive cell population. The anti-OX40 antibody non-reactive cell population undergoes the final single cell sorting step. After several days of cell expansion, flow cytometry was used OX40 antibody and non-binding monoclonal antibody pab1949-1 or pab1928 were tested again for single-cell sorted cells. In short, 1624-5 cells expressing individual human OX40 alanine mutants were cultured with monoclonal anti-OX40 antibodies pab1949-1 or pab1928. For each antibody, two antibody concentrations were tested (pab1949-1: 2 μg/ml and 0.5 μg/ml; pab1928: 1.1 μg/ml and 0.4 μg/ml). Multiple anti-OX40 antibodies (AF3388, R&D systems) conjugated to APC were diluted 1:2000. The FC receptor block (1:200; BD catalog number 553142) was added, and the samples were incubated for 20 minutes at 4°C. After washing, if necessary for detection (PE-conjugated; BD catalog number 109-116-097), the cells were incubated with a second anti-IgG antibody for 20 min at 4°C. Then, the cells were washed and collected using a flow cytometer (BD Biosciences).

In order to link the phenotype (multiple strains of anti-OX40 antibody+, single strain of anti-OX40 antibody-) with the genotype, the single-cell sorted human OX40 mutants were sequenced. Figure 21 shows a table of human OX40 alanine mutants that still bind to multiple anti-OX40 antibodies but do not bind to the monoclonal anti-OX40 antibodies pab1949-1 or pab1928. All residues are numbered according to the mature amino acid sequence of human OX40 (SEQ ID NO: 55). Based on flow cytometry analysis, "+" means binding, and "-" means lack of binding.

The present invention is not limited to the specific embodiments described here Set range. Indeed, from the above description and drawings, in addition to the content described, the various modifications of the present invention will also become clear to those who are familiar with the art. Such changes are intended to fall within the scope of the attached patent application.

For all purposes, all references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in their entirety, and this achieves the same as each individual reference (e.g., publication or (Patents or patent applications) specifically or individually indicate the same extent to which they are incorporated by reference in their entirety for all purposes.

Other embodiments are within the scope of the following patent applications.

Claims (22)

An isolated antibody that specifically binds to human OX40, wherein the binding between the antibody and the OX40 variant is substantially weakened relative to the binding between the antibody and the human OX40 sequence of SEQ ID NO: 55, and wherein the OX40 variant Containing the sequence of SEQ ID NO: 55, only amino acid mutations selected from the group consisting of N60A, R62A, R80A, L88A, P93A, P99A, P115A and their combinations, wherein the antibody does not contain SEQ ID NO: 4 VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the amino acid sequences of, 5, 6, 1, 2, and 3. An isolated antibody that specifically binds to human OX40, wherein the binding between the antibody and the OX40 variant is substantially weakened relative to the binding between the antibody and the human OX40 sequence of SEQ ID NO: 55, and wherein the OX40 variant Containing the sequence of SEQ ID NO: 55, only amino acid mutations of W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A exist, wherein the antibody does not contain SEQ ID NO: 4, 5, 6, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the amino acid sequences of 1, 2, and 3. An isolated antibody that specifically binds to human OX40, in which compared to binding to the human OX40 sequence of SEQ ID NO: 55, the antibody exhibits reduced or no binding to the following protein: the same as SEQ ID NO: 55 Protein, there are only amino acid mutations selected from the group consisting of N60A, R62A, R80A, L88A, P93A, P99A, P115A and their combinations, wherein the antibody does not contain SEQ ID NO: 4, 5, 6. VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the amino acid sequence of 1, 2, and 3. An isolated antibody that specifically binds to human OX40, in which compared to binding to the human OX40 sequence of SEQ ID NO: 55, the antibody exhibits reduced or no binding to the following protein: the same as SEQ ID NO: 55 Proteins, only amino acid mutations of W58A, N60A, R62A, R80A, L88A, P93A, P99A and P115A exist, where the antibody does not contain the amines comprising SEQ ID NOs: 4, 5, 6, 1, 2 and 3, respectively The base acid sequence of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3. An isolated antibody that specifically binds to human OX40, wherein the antibody specifically binds to an epitope of the human OX40 sequence, the epitope comprising residues 60, 62, 80, 88, 93, 99, of SEQ ID NO: 55, 115 and the residues of their combination, wherein the antibody does not include the VH-CDR1, VH-CDR2, VH-CDR3, VH-CDR3, VH-CDR3, VH-CDR3, VH-CDR3, VH-CDR3, VH-CDR3, VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2 and VL-CDR3. An isolated antibody that specifically binds to human OX40, wherein the antibody specifically binds to an epitope of the human OX40 sequence, the epitope comprising residues 58, 60, 62, 80, 88, 93, 99 and 115, wherein the antibody does not contain the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and the amino acid sequence comprising SEQ ID NO: 4, 5, 6, 1, 2 and 3, respectively VL-CDR3. An isolated antibody that specifically binds to human OX40 Body, wherein the antibody specifically binds to at least one residue of SEQ ID NO: 55, and the at least one residue is selected from residues 60, 62, 80, 88, 93, 99, 115 and combinations thereof, wherein the antibody VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, which include amino acid sequences of SEQ ID NOs: 4, 5, 6, 1, 2, and 3, respectively, are not included. An antibody according to any one of the preceding claims, wherein the antibody comprises a human immunoglobulin IgG ₁ heavy chain constant region, and wherein the _{amino acid sequence of the IgG 1} heavy chain constant region comprises selected from the group consisting of N297A, N297Q, D265A and others A combination of mutations or mutations selected from D265A, P329A, and a combination of them. An isolated antibody that specifically binds to human OX40, the antibody comprising the heavy chain variable region sequence and the light chain variable region sequence of the antibody according to any one of the preceding claims, wherein the antibody is selected from the group consisting of : Fab, Fab', F(ab') ₂ and scFv fragments. An isolated antibody that specifically binds to human OX40, the antibody comprising a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise the heavy chain variable region sequence and the light chain of the antibody according to any one of the preceding claims. Variable region sequence. The antibody of claim 10, which further comprises a human immunoglobulin IgG ₁ heavy chain constant region, wherein the _{amino acid sequence of the IgG 1} heavy chain constant region comprises a mutation selected from the group consisting of N297A, N297Q, D265A and their combination Or a mutation selected from D265A, P329A and their combination. The antibody of any one of the preceding claims, wherein: (a) The antibody is antagonistic to human OX40; (b) The antibody inactivates, reduces or inhibits the activity of human OX40; (c) The antibody inhibits or reduces the binding of human OX40 to the ligand of human OX40; (d) The antibody inhibits or reduces human OX40 transmission; (e) The antibody inhibits or reduces human OX40 signaling induced by human OX40 ligand; or (f) The antibody further includes a detectable label. An isolated nucleic acid molecule that encodes: (a) The heavy chain variable region or heavy chain of an antibody according to any one of claims 1 to 12; (b) The light chain variable region or light chain of an antibody according to any one of claims 1 to 12; or (c) The heavy chain variable region or heavy chain of the antibody according to any one of claims 1 to 12 and the light chain variable region or light chain of the antibody according to any one of claims 1 to 12. An isolated vector comprising the nucleic acid molecule of claim 13. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14. A pharmaceutical composition comprising an antibody as claimed in any one of claims 1 to 12, a nucleic acid molecule as claimed in claim 13, a vector as claimed in claim 14 or a host cell as claimed in claim 15 and pharmaceutically acceptable Of excipients. A method for producing an antibody that binds to human OX40, the method comprising culturing a host cell as in claim 15 so that the nucleic acid molecule can be expressed and the antibody can be produced. Use of an antibody according to any one of claims 1 to 12, a nucleic acid molecule as claimed in claim 13, a vector as claimed in claim 14, a host cell as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16 in the preparation of a medicine , The drug is used for: (a) Regulate the individual's immune response; (b) Regulating the immune response of the individual, wherein regulating the immune response includes enhancing or inducing the immune response of the individual; (c) Regulating the immune response of the individual, wherein regulating the immune response comprises reducing or suppressing the immune response of the individual; (d) Enhance individual T cell expansion and T cell effector function; (e) Treating the individual's cancer; or (f) Treating cancer in an individual, wherein the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, colon cancer, and lung cancer. Such as the purpose of claim 18, where: (a) The drug further contains an inhibitor of indoleamine-2,3-dioxygenase (IDO); (b) The drug further comprises an inhibitor of indoleamine-2,3-dioxygenase (IDO), wherein the inhibitor is selected from the group consisting of: epacadostat, F001287 , Indoximod and NLG919; (c) The medicine further contains a vaccine; or (d) The medicine further comprises a vaccine, wherein the vaccine comprises a heat shock protein peptide complex (HSPPC), and the HSPPC comprises a heat shock protein complexed with an antigen peptide, wherein (i) The heat shock protein is a gp96 protein and is complexed with a tumor-associated antigen peptide, wherein the HSPPC is derived from a tumor obtained from the individual, or (ii) The heat shock protein is hsp70 or hsc70 protein and is complexed with tumor-associated antigen peptides. Such as the use of claim 18 or 19, where the system is human. A method for detecting OX40 in a sample, the method comprising contacting the sample with the antibody according to any one of claims 1-12. A kit comprising the antibody of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the vector of claim 14, the host cell of claim 15, or the medicine of claim 16 Composition and a) detection reagent, b) OX40 antigen or c) a combination of them.

Images